We conclude that TRAFD1, in association with Plekhm1/Rab7-positive late endosomes-early lysosomes, has a previously unknown role in vesicle trafficking, acidification, and resorption in osteoclasts.Plekhm1 is a large, multi-modular, adapter protein implicated in osteoclast vesicle trafficking and bone resorption. In patients, inactivating mutations cause osteopetrosis, and gain-of-function mutations cause osteopenia. Investigations of potential Plekhm1 interaction partners by mass spectrometry identified TRAFD1 (FLN29), a protein previously shown to suppress toll-like receptor signaling in monocytes/macrophages, thereby dampening inflammatory responses to innate immunity. We mapped the binding domains to the TRAFD1 zinc finger (aa 37-60), and to the region of Plekhm1 between its second pleckstrin homology domain and its C1 domain (aa 784-986). RANKL slightly increased TRAFD1 levels, particularly in primary osteoclasts, and the co-localization of TRAFD1 with Plekhm1 also increased with RANKL treatment. Stable knockdown of TRAFD1 in RAW 264.7 cells inhibited resorption activity proportionally to the degree of knockdown, and inhibited acidification. The lack of acidification occurred despite the presence of osteoclast acidification factors including carbonic anhydrase II, a3-V-ATPase, and the ClC7 chloride channel. Secretion of TRAP and cathepsin K were also markedly inhibited in knockdown cells. Truncated Plekhm1 in  Bone development, remodeling and repair are carried out by osteoblasts which produce bone matrix, osteocytes which communicate the conditions inside the bones, and osteoclasts which resorb bone . NormallOsteoclast formation from bone marrow precursors is a main point of control in bone resorption, and RANKL and M-CSF are required to induce expression of genes that typify the osteoclast . RANKL bMembrane dynamics are of central importance in osteoclasts –13. DiffSNX10 account for roughly 4% of ARO in humans, often including an osteopetro-rickets phenotype, and Snx10 is required for osteoclast differentiation and function [ia) rat when truncated[The importance of intracellular vesicle trafficking was emphasized with the discovery of two proteins involved in that process which cause osteoclast bone disease when mutated. One is the sorting nexin, Snx10. Snx10 regulates endosome sorting and movement through the cell. Mutations in function –19. The truncated, and whitruncated. Plekhm1truncated. During TRAFD1 was first identified as an interferon- (IFN) and lipopolysaccharide- (LPS) inducible factor . TRAFD1 ia/ia osteopetrotic rat osteoclasts which are unable to resorb bone, truncated Plekhm1 prevented TRAFD1 association with vesicles.In the present study, we confirmed and mapped in detail the interaction of TRAFD1 and Plekhm1 We also showed that TRAFD1 expression is up-regulated by RANKL, that it co-localizes with Plekhm1 to late endosomes, and that co-localization of TRAFD1 and Plekhm1 increases after RANKL stimulation. We also show here that TRAFD1 affects osteoclast acidification, mineral solubilization, and secretion of tartrate-resistant acid phosphatase (TRAP) and cathepsin K. In ia rats and C57BL/6J mice maintained at the University of Massachusetts Medical School under specific-pathogen-free conditions, and all procedures were in accordance with the NIH Guide for the Care and Use of Laboratory animals and were approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. Euthanasia was performed by inhalation anesthesia followed by decapitation.All animals were obtained from our colonies of The following antibodies were used in the study: rat anti-HA , mouse anti-FLAG M2 , rabbit anti-lamin B1 . Goat anti-Rab7 (# sc-6563), goat anti-ClC7 (# sc-16442), and mouse anti-CAII (# sc-166569) were purchased in Santa Cruz Biotechnology . Rabbit anti-Rab7 (# 2094) and rabbit anti-NFAT2 (# 8032) were purchased from Cell Signaling Technology . Rabbit anti-cathepsin K was a gift from Dr. Eunice Lee, Shriners Hospital for Children, Montreal, Canada; rabbit anti-TRAP was a gift from Dr. Göran Andersson, Karolinska Institutet, Stockholm, Sweden; rabbit anti-a3-vATPase was a gift from Dr. Beth Lee, Ohio State University, Columbus, OH. Anti-Plekhm1 antibody was raised in rabbits against the N-terminal 497 amino acids of human Plekhm1 protein using a standard immunization protocol , and the antiserum was affinity purified against the immunizing protein using UltraLink Biosupport resin . Anti-TRAFD1 antibody was raised in chicken against whole affinity purified human TRAFD1 protein using a standard immunization protocol . IgY was precipitated from egg yolk and affinity purified with the immunizing antigen using Ultra Link Iodoacetyl Gel resin (Thermo Scientific) according to the manufacturer’s protocol. Secondary HRP-conjugated antibodies used for immunoblotting were: goat anti-rabbit IgG and rabbit anti-goat IgG , sheep anti-mouse IgG , and donkey anti-chicken IgY . Secondary fluorescent dye-conjugated antibodies used for immunofluorescence: donkey anti-chicken IgY, Alexa 488 and TRITC (#703-545-155 and 703-025-155), donkey anti-goat IgG, Alexa 488 (# 705-486-147), and donkey anti-rabbit IgG, Alexa 568 (#711-295-152), purchased from Jackson ImmunoResearch Laboratories Inc. . Donkey anti-rabbit IgG, Alexa 488 (#A21206) was from Life Technologies .2. Bone marrow mononuclear cells (BMMC) and splenocytes were collected from mice (C57BL/6J) and rats (ia strain) as described previously [RAW264.7 and HEK 293 cells  were maintained in α-minimum essential medium (α-MEM) or Dulbecco’s modified Eagle's medium (DMEM), respectively, supplemented with 10% FBS, 1% penicillin/streptomycin in a humidified incubator at 37°C in 5% COeviously ,25. ThesRplp0) [TRAFD1-targeting shRNA in pLKO.1 vector was obtained from Sigma Aldrich (SHCLND-NM_006700). shRNA sequences were as follows: shRNA 1: GGCTTGAGTGTGCAGCGTGTTACCTCGAGGTAACACGCTGCACACTCAAGTTTTTG (TRCN0000339582); shRNA2: CGGCATGCCTTACGTTCACTCAATCTCGAGATTGAGTGAACGTAAGGCATGTTTTTG (TRCN0000339580); shRNA3: CGGCACCTACTCGATGTCTCCTAACTCGAGTTAGGAGACATCGAGTAGGTGTTTTTG (TRCN0000339509); shRNA4: CGGCGCACACTTGGACTTCATGTTCTCGAGAACATGAAGTCCAAGTGTGCGTTTTTG (TRCN0000339508). The scrambled shRNA control sequence was: CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG, generated by Sarbassov et al.  and obtaRplp0) .8) stably expressing low levels of TAP-tagged Plekhm1 was incubated overnight at 4°C with 100 μl packed volume of Streptavidin Sepharose . Streptavidin beads were washed and protein complexes were eluted from the streptavidin resin in calmodulin binding buffer supplemented with 50 mM biotin. The second round of affinity purification was performed using 100 μl of Calmodulin Sepharose . After washing, the protein complexes were eluted twice with 100 μl wash buffer containing EGTA. The eluates were combined, run briefly into an SDS-PAGE gel, excised, and sent to the Proteomic and Mass Spectrometry Facility at University of Massachusetts Medical School to be digested with trypsin and analyzed by LC-MS/MS.The pIRES-puro Glue vector (TAP tagged), generated by Angers et al., , was pur3VO4) supplemented with fresh 2 mM DTT and protease inhibitor cocktail (Sigma Aldrich). Lysates were cleared by centrifugation and pull-downs were performed using 25 μl of Streptavidin Sepharose for 2 hours at 4°C. After extensive washing of beads with lysis buffer, precipitated proteins were detected by immunoblot analysis using chemiluminescent detection. For immunoprecipitation of FLAG-tagged TRAFD1, HEK293 cells were lysed with lysis buffer . 15 μl of FLAG-M2 gel (Sigma Aldrich) was incubated with cell lysate for 2 hours at 4 C and pellets were washed 5 times with wash buffer  prior to loading onto SDS-PAGE gels for analysis.Full-length human cDNA for Plekhm1 and various deletion mutants were PCR-amplified and cloned into the pIRES-puro Glue vector downstream of the tandem affinity tags. The p3×FLAG-CMV14 plasmid with human cDNA for TRAFD1 was a gift from Dr. Akihiko Yoshimura, Keio University, Tokyo, Japan). Deletion mutants of the cDNA were PCR-amplified and cloned into the p3×FLAG-CMV14. A 6 cm dish of HEK293T cells was transfected with p3xFLAG-CMV14-TRAFD1 and pIRES-puro Glue–Plekhm1, either full-length or deletion mutants using Lipofectamine 2000 (LifeTechnologies). 24 hours post-transfection, the cells were washed with ice-cold PBS twice and lysed in 0.5 ml of lysis buffer , rabbit anti-Plekhm1 (1:1000), goat anti-Rab7 (1:100). All were incubated for 1 hour at room temperature and rinsed. Fluorescent dye-conjugated secondary antibodies were then incubated for 45 minutes. DAPI (Life Technologies) staining was used to label nuclei. The samples were mounted with ProLong Gold antifade reagent (Life Technologies) and observed with a Leica SP5 (II) laser scanning confocal microscope  equipped with 40 × (1.30 NA) and 63 × (1.4–0.6 NA) oil immersion lenses. Leica LAS AF Lite software was used for recording and image processing.260. 1 μg of total RNA was subjected to reverse transcription with a QuantiTect Reverse Transcription Kit (Qiagen). The resulting cDNA was used for PCR using a QuantiFast SYBR Green PCR kit (Qiagen). Amplification reactions were performed in triplicate in 10 μl final volume that included the following: 10–50 ng of template, 1 μM primers, 2× SYBR Green Master Mix. Reactions were processed in a LightCycler 2 (Roche Diagnostics): 95°C for 5 min, then 40 cycles of 95°C for 10 s and 60°C for 30 s. ΔCt for each gene was calculated and represents the difference between the Ct value for the gene of interest and that of the reference gene. Fold-changes were calculated using the 2−ΔΔCt convention [RNA was extracted using RNeasy  and the yield determined by measuring ODnvention . Primer Cells at a density 15,000/well were cultured for 10 days on Osteo Assay Surface (hydroxyapatite) 24-well plates  under differentiation conditions. To observe resorption pits, plates were gently stripped of cells with 10% bleach, rinsed with distilled water, air dried, and scanned on a flatbed scanner (Microtek 9800 XL). The percentage of resorbed area was analyzed by NIH ImageJ software.Fixed cells were TRAP-stained using a leukocyte acid phosphatase kit (Sigma Aldrich) according to the manufacturer’s instructions. TRAP-positive multinucleated cells containing three or more nuclei were counted.RAW264.7 cells were cultured in α-MEM containing RANKL on HA plates until differentiated (3–4 days). On the day when cells differentiated, the medium was changed. Conditioned medium was collected on subsequent days 2 and 5 for measurement of TRAP activity as described previously . AbsorbaRAW264.7 cells were differentiated on HA plates, as above. When cells differentiated, the medium was changed, and at the time points indicated, conditioned medium was collected. Cells from each well were also collected and total cell protein extracts were prepared by lysis in RIPA buffer. Supernatant (40 ul) and total cell lysate (50 ug) were resolved on SDS-PAGE gels, blotted onto PVDF, and probed with anti-cathespin K. Blots were subsequently probed with anti-lamin B1 antibody as a loading control.RAW264.7 cells were cultured for 5 days on Osteo Assay 24-well plates under differentiation conditions. Acridine orange  . As a coia/ia rats were grown in 150 mm dishes in the presence of RANKL (20 ng/ml) until differentiated (4 days). The cell fractionation protocol was adapted from [6) were washed twice with PBS and scraped into hypo-osmotic buffer . After 5 minutes on ice, cells were homogenized by 10 passes through a 23Ga needle attached to a 1 ml syringe and diluted with an equal volume of hyper-osmotic buffer . The cell lysate was then centrifuged at 1000 Gav for 10 minutes at 4°C. The nuclear pellet from this centrifugation was resuspended in RIPA buffer . The post-nuclear supernatant was collected into a new tube and centrifuged at 15,000 Gav for 15 minutes at 4°C. The pelleted light mitochondrial fraction (LMF) was resuspended in RIPA buffer and the post-mitochondrial supernatant was further centrifuged at 100,000 Gav for 60 minutes at 4°C. The pelleted vesicle fraction was resuspended in RIPA buffer and the remaining supernatant was saved as the cytosolic fraction. Protein fractions were frozen at -80°C until analyzed. Nuclear and cytosolic cell extracts were isolated according Kloet et al. [Mononuclear cells isolated from wild-type (WT) and ted from  with modt et al. .+ Imaging System (Bio Rad).Protein concentrations of whole cell extracts were determined using Coomassie Plus Protein Assay Reagent (Thermo Scientific) according to the manufacturer’s instructions. 25–150 μg of total protein was loaded per lane for SDS-PAGE. The gels were blotted onto PVDF membranes and probed with the following primary antibodies at the indicated dilutions: 1:10,000 Plekhm1; 1:250 TRAFD1; 1:1000 Rab7 ; 1:1000 CAII, 1:2000 cathepsin K; 1:2000 TRAP; 1:3000 a3-vATPase; 1:1000 HA; 1:5000 FLAG M2; 1:500 α-tubulin; and 1:5000 lamin B1. The appropriate horseradish peroxidase-conjugated secondary antibody was used at 1:2000 to 1:5000 dilution. Proteins of interest were visualized with ECL substrate either by film exposure (Thermo Scientific) or Gel Doc XRt-test (Microsoft Excel or GraphPad Prism 6) was used for statistical comparisons of two groups, while one-way ANOVA (GraphPad Prism 6) was used to compare more than two groups. For all analyses a P-value of less than 0.05 was considered to be statistically significant.All quantitative data are shown as mean + s.d. of at least three (n = 3) biological replicates for each experiment. Two-tailed Student's . Values obtained were close to zero, validating the positive co-localizations in the non-rotated images [Co-localization analysis was done using Just Another Co-localisation Plugin (JACoP) for Image J (NIH), in which Pearson’s correlation coefficient values were analyzed. A value of 1 represents perfect correlation, while a value close to 0 represents random correlation. To eliminate the possibility of false-positives, one of the images was rotated 90° and co-localization was measured again d images .Fig 1A, lower panel) were tested for their ability to interact with full-length Plekhm1, and the reciprocal experiments were performed with full-length TRAFD1 and deletion constructs of Plekhm1 . Both full-length TRAFD1 and a TRAFD1 N-terminal deletion mutant (aa Δ1–37) bound full-length Plekhm1 . Further truncations of TRAFD1 prevented binding to Plekhm1. The interacting N-terminal region of TRAFD1 is comprised of amino acids 37–60 and is part of the zinc finger domain (28–103 aa). Both full-length Plekhm1 and a more C-terminal region of Plekhm1 (778–986 aa) were immunoprecipitated with FLAG-tagged TRAFD1. Together, these results demonstrated that Plekhm1 and TRAFD1 directly interact and that the zinc finger domain of TRAFD1 is necessary to bind to Plekhm1 between its second PH domain and the C1 domain.Plekhm1 is a large, modular cytoplasmic protein localized to late endosomes/early lysosomes and involved in vesicle transport and ruffled border formation in osteoclasts. Previously, we and others identified mutations of Plekhm1 that cause osteopetrosis in rats and humans  and osteFig 2A). Western blot analysis demonstrated that TRAFD1 protein is expressed at a low level in RAW264.7 cells and BMMC, and that RANKL caused increases in TRAFD1 protein in both cell types , suggesting an increase in protein stability, at least in primary cells.Previously , it was Fig 3A). The co-localization further increased when BMMC-derived osteoclasts were actively resorbing on HA plates. Pearson’s correlation analysis showed that co-localization of TRAFD1 and Plekhm1 increased from 0.59±0.05 in BMMC to 0.72±0.07 and 0.80±0.04 in osteoclasts and resorbing osteoclasts, respectively (P<0.0001 for monocytes vs. osteoclasts and P<0.0001 for monocytes vs. resorbing osteoclasts). Previously [Fig 3B). This confirmed that Plekhm1 strongly co-localizes with Rab7 to late-endosomal/early-lysosomal vesicles in mononuclear cells (Pearson’s coefficient = 0.63±0.11) and the level of co-localization does not change in mature osteoclasts (Pearson’s coefficient = 0.66±0.05). Control analysis using 90° rotation of color channel images [vs. 0.71± 0.06 in osteoclasts) places a substantial fraction of TRAFD1 in the same cellular functional compartment as Plekhm1, i.e., late endosomes/early lysosomes of monocytes and differentiated osteoclasts .In order to investigate interactions between TRAFD1 and Plekhm1 in osteoclasts, we analyzed co-localization in BMMC by immunofluorescence. We found an increase in co-localization upon RANKL stimulation . When the clones were cultured with RANKL on Osteo Assay Plates  for 10 days, defects in resorbing ability were found that reflected the degree of TRAFD1 knockdown . We used the clone with the lowest TRAFD1 level and the lowest resorption ability, 2.6, designated hereafter as “shTRAFD1,” in subsequent experiments.In order to investigate potential functions of TRAFD1 in osteoclasts, RAW264.7 cells were transduced with lentivirus encoding shTRAFD1, and stable clones expressing different levels of TRAFD1 mRNA were selected , shTRAFD1 cells did form multinucleated, TRAP-positive cells . Because we observed a delay in differentiation of shTRAFD1cells, we examined the status of the main osteoclast transcription factor NFATc1. In control cells, we observed increased total NFATc1 protein 24 hours after adding RANKL, and the expression increased until day 3. In shTRAFD1 cells, the NFATc1 level was slightly lower and the translocation of NFATc1 into the nucleus was delayed compare to the control cells, consistent with the delayed differentiation of shTRAFD1 cells .Due to the lack of resorption of HA by shTRAFD1 cells, we investigated their capacity to differentiate and to express factors required for resorptive activity. As shown in Fig 5B). This prompted us to investigate whether the impaired resorption was the result of reduced levels of known acidification and resorption factors, including the ClC7 chloride channel, cathepsin K, matrix metallopeptidase 9 (MMP-9), carbonic anhydrase II and the osteoclast-specific subunit of the vacuolar proton pump, a3-vATPAse . To this end, we measured gene expression levels by Q-PCR and also evaluated western blots of differentiated cells. At the mRNA level, TRAP, MMP-9 and carbonic anhydrase II were significantly reduced  . At the protein level, only cathepsin K showed a reduction. All the other proteins assayed appeared to be present at normal levels. We also observed that differentiation of shTRAFD1 cells with RANKL caused an increase in the level of TRAFD1 mRNA, but we did not observe any changes in the protein level.Further, those cells exhibited the actin rings characteristic of osteoclasts, as shown by fluorescent labeling with phalloidin . In this assay, osteoclast-like cells with normal acidification display orange-red staining, indicating active production of H+ [Since actin rings were present in shTRAFD1 cells, and protein levels of resorption factors (with the lone exception of cathepsin K) were normal, we investigated whether acidification was occurring properly in shTRAFD1 cells. Cells were differentiated by culturing with RANKL for 5 days on HA plates, and then the live cells were labeled with the acidotropic probe acridine orange . On day 2, TRAP activity in conditioned medium from shTRAFD1 cells was 12-fold lower than in control cell medium . Also, as shown in Fig 8B, shTRAFD1 cells secreted markedly less cathepsin K than controls. These data suggest that TRAFD1 is important for targeting cathepsin K and TRAP to their correct location at the ruffled border and are consistent with a role for TRAFD1 in osteoclast vesicle transport and secretion.We investigated a possible role for TRAFD1 in osteoclast exocytosis by analysis of TRAP and cathepsin K secretion. TRAP activity was measured biochemically, and cathepsin K was analyzed by western blotting (ia/ia rats (which lack bone marrow spaces). Immunoblot analysis of the resulting fractions is shown in Fig 9. In WT osteoclasts, most of the Plekhm1 was present in the vesicle fraction (lane VF) and the cytosolic fraction (lane CF). The vesicle fraction in these separations contains endosomes and early lysosomes. Organelle-specific antibody for late endosomes/early lysosomes (Rab7) confirmed the enrichment of those vesicles in this fraction in WT osteoclasts. Unlike Plekhm1, no Rab7 was detected in the cytosolic fraction. TRAFD1 appeared roughly equally partitioned between the light mitochondrial fraction (LMF) lane, the VF lane, and the CF lane. In osteoclasts generated from ia/ia rat cells, we observed that their truncated form of Plekhm1 was primarily present mainly in the heavy nuclear pellet (lane NP) and the CF lane, and was absent from the vesicle fraction. This mirrored the altered distribution of Rab7. TRAFD1 in ia/ia osteoclasts was detected mostly in the LMF lane; however, with longer exposure we detected a small amount of TRAFD1 in the CF lane and the VF lane. Together, these results suggest that Plekhm1 truncation disrupts both the stability and the vesicular localization of TRAFD1.To further define the association of TRAFD1 with osteoclast vesicles, we performed sequential ultracentrifugal fractionation of osteoclasts generated from bone marrow cells of WT rats and splenocytes from TRAFD1, also known as FLN29, was previously linked to signaling by toll-like receptors (TLR) and retinoic acid-inducible gene 1 (RIG-1)-like helicase (RLH), dampening the inflammatory response in macrophages ,24. In tThe level of TRAFD1 protein in osteoclast precursors was relatively low, although it increased during RANKL stimulation. We previously observed a similar pattern for Plekhm1 . Confocaia/ia osteopetrotic rat cells, which have a truncation at codon 338 of Plekhm1 , TRAFD1/Rab7/Plekhm1 interactions were disrupted. The distribution of those proteins in sequential centrifugation fractions was altered. In WT osteoclasts, Plekhm1 mainly co-purified with the vesicle fraction, and similar purification was observed for Rab7 and TRAFD1. The truncated ia/ia Plekhm1, however, did not co-fractionate with Rab7, and the TRAFD1 level was markedly reduced in the cell lysate and in the vesicle fraction. This suggests that the Plekhm1/Rab7/TRAFD1 interaction is important for the resorbing and secretory activities of osteoclasts and for TRAFD1 stability.The amino acids of TRAFD1 which bind to Plekhm1, 37–60, are part of a TRAF-like zinc finger domain. That domain interacts directly with amino acids 778–986  of Plekhm1. That region of Plekhm1 lies in a conserved sequence between the second of 2 pleckstrin homology (PH) domains and the C1/ZnF domain near the C-terminus. Those are both required for proper interaction with the small GTPase Rab7 ,37. Rab7Rab7 and Plekhm1 regulate the secretory lysosomal pathway, which delivers cathepsin K and acidic vesicles to the ruffled border . Plekhm1ia/ia rat truncation; see S4 Fig) or gain-of-function (R714C in an osteopenic patient) mutations in Plekhm1 [ia/ia rat osteoclasts  suggests blockage of delivery of cargo to the ruffled border, and might explain both the pelleting of Rab7 with the relatively heavy mitochondrial fraction and the lack of ruffled borders in ia/ia rat osteoclasts [Interestingly, disruption of dynein-dynactin complexes in resorbing osteoclasts did not impair acidification, but did cause a defect in cathepsin K secretion through the ruffled border . Another Plekhm1 . The slieoclasts . TogetheFrom the results described above, we conclude: 1) TRAFD1 interacts specifically with Plekhm1; 2) that it is required for establishment of effective acidification of the resorption lacuna; 3) that it is significantly associated with Rab7-positive vesicles; and 4) that it is important in the secretory activity of resorbing osteoclasts. The importance of a clearer mechanistic understanding of cathepsin K secretion is underscored by the ongoing development and trials of a cathepsin K inhibitor (odanacatib) to prevent bone loss and tumor metastasis to bone . CremascS1 Fig(A). As a control and to exclude false-positive results, one of the images were rotated 90°C and the co-localization was measured again (B).Co-localization analysis was done using JACoP plugin for Image J. Pearson’s correlation coefficient values were analyzed using green and red channel of each image (TIF)Click here for additional data file.S2 Figvs. 0.71± 0.06 in osteoclasts). Student t-test was carried out and values are mean + s.d. of n = 3 independent experiments analyzing at least 4 images/condition. **P<0.001 versus monocytes. Mo = monocytes; OCs = osteoclasts.Immunostaining for endogenous TRAFD1 and Rab7 was performed on mouse monocytes and on mouse monocytes treated with RANKL cultured on glass (osteoclasts). Confocal microscopy images were obtained using anti-Rab7 (green) and anti-TRAFD1 (red), and representative images are shown. DAPI staining was used to visualize nuclei. Scale bars = 10 μm. Insets show enlarged regions outlined in white. Arrowheads indicate examples of vesicles where co-localization gives yellow signal. Pearson’s correlation coefficient was used to estimate the co-localization of TRAFD1 with Rab7 (Pearson’s coefficient = 0.56±0.11 in monocytes (TIF)Click here for additional data file.S3 Fig(A) Western blot analysis of expression levels of NFATc1 in total cell extracts from control and shTRAFD1 cells cultured in the presence of RANKL for 1–3 days. Cells were lysed, blotted, and probed with antibody to NFATc1. α-tubulin was used as a loading control, as indicated. (B) Western blot analysis of expression levels of NFATc1 in nuclear and cytosolic fractions of control and shTRAFD1 cells stimulated with RANKL for 1–3 days.(TIF)Click here for additional data file.S4 Figia/ia (splenocytes) rats. Cells were cultured on 24-well Osteo Assay plates for 5 days in the presence of RANKL (20 ng/ml) and stained with acridine orange. Representative confocal microscopy images are shown. As a control for blocked acidification, some control cells were treated with bafilomycin A . Scale bar = 50 μm. Virtually all the orange staining in the ia/ia cells was in large vesicles, whereas in WT cells, there was staining spread out, presumably under the ruffled borders, which are absent from ia/ia rat osteoclasts.Acidification assay was performed on cells from 7-day-old WT (BMMC) and (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


FLT3, ABCB1, VEGFR2, ABCG2 and BIM with patient toxicities, response, and survival. We observed a stronger association of FLT3 738T genotype with leucopenia in our Asian dataset than that previously reported in Caucasian mRCC patients . We observed significant associations of FLT3 738T (OR=2.7), ABCB1 1236T (OR=0.3), ABCB1 3435T (OR=0.1), ABCB1 2677T (OR=0.4), ABCG2 421A (OR=0.3) alleles and ABCB1 3435, 1236, 2677 TTT haplotype (OR=0.1) on neutropenia. Primary resistance  and inferior survival  were associated with the ABCB1 3435, 1236, 2677 TTT haplotype. In conclusion, ABCB1 and FLT3 polymorphisms may be helpful in predicting sunitinib toxicities, response and survival benefit in Asian mRCC patients. We have also validated the association between FLT3 738T and sunitinib-induced leucopenia previously reported in Caucasian populations, but have not validated other reported genetic associations.Sunitinib is a tyrosine kinase inhibitor used as first-line treatment for metastatic renal cell carcinoma (mRCC). Asian ethnicity has been previously associated with lower clearance and greater toxicities for sunitinib treatment, relative to Caucasian ethnicity. Research focusing on identifying corresponding biomarkers of efficacy and toxicity has been hitherto conducted in Caucasian populations, and few of the reported associations have been externally validated. Our work thus aims to investigate candidate biomarkers in Asian patients receiving sunitinib, comparing the observed genotype effects with those reported in Caucasian populations. Using data from 97 Asian mRCC patients treated with sunitinib, we correlated 7 polymorphisms in  Sunitinib is a tyrosine kinase inhibitor that targets vascular endothelial growth factor receptors , platelet-derived growth factors (PDGFRα and PDGFβ), Fms-like tyrosine kinase 3 (FLT3) and the RET protein . It is uVEGFR1 [VEGFR2 [PDGFRα [FLT3 [FLT4 [VEGFA [FGFR2 [eNOS [CYP3A5 [CYP1A1 [NR1/3 [NR1/2 [ABCG2 [ABCB1 [VEGFR3 3971 GG genotype and better progression-free survival in two independent studies [Since the FDA approval of sunitinib in 2006, genetic biomarkers have received intense research attention as a promising measure to personalize sunitinib use by profiling individual toxicity and response predisposition. To this end, several prior studies  have corVEGFR1 , VEGFR2  [VEGFR2 , 12, PDG [PDGFRα , FLT3 [1Rα [FLT3  and FLT4T3 [FLT4 , 14), ot4 [VEGFA , FGFR2 [A [FGFR2  and eNOSR2 [eNOS ), hepati [CYP3A5  and CYP1 [CYP1A1 , 19), he1 [NR1/3  and NR1/3 [NR1/2 ), as wel2 [ABCG2 , 20 and 2 [ABCB1 , 11, 21. studies , 18. The1236C/T, 3435C/T and 2677G/T of ABCB1 for their demonstrated effect on the functionality of the multi-specificity transporter encoded [ABCG2; brain accumulation of sunitinib has been observed to increase significantly in ABCB1 knockout and ABCB1/ABCG2 double knockout mice, despite bioavailability after oral dosing remaining similar to that of wild type mice [ABCB1 1236C/T and 2677G/T were found to be associated with the clearance of sunitinib in a study involving 114 cancer patients in the Netherlands [ABCB1 polymorphisms have been associated with hand-foot syndrome and survival in sunitinib receivers in exploratory studies [ABCB1 1236 T allele was found in 71.9% and 41% of a Chinese [ABCB1 polymorphisms with sunitinib treatment outcomes in Asian patients.We identified three candidate polymorphisms,  encoded , 23. Sunype mice . Recentlherlands . Current studies , 19 in E Chinese  respectiBIM gene was found to be associated with unfavorable outcomes upon treatment with multiple tyrosine kinase inhibitors (TKIs). For example, inferior imatinib response in chronic myelogenous leukemia and shorter progression-free survival in EGFR—mutated non—small-cell lung cancer treated with gefitinib or erlotinib was observed in an Asian population [BIM in sunitinib activity has been suggested by several prior animal and in vitro studies—Naik et al. demonstrated that destruction of tumor vasculature by VEGF-blocking antibodies was BIM-dependent [et al. noted that there was upregulation of BIM along with other proapoptotic genes in human medulloblastoma cell lines treated with sunitinib [BIM deletion with outcomes in sunitinib-receiving patients is therefore a subject of interest to us.Recently, a 2,903-base-pair deletion polymorphism in intron 2 of the pulation . The likpulation . The invependent  and Yangunitinib . The invThis study aimed to evaluate genetic polymorphisms to investigate their association with sunitinib toxicities and survival benefits in Asian renal cancer patients. We have selected candidate polymorphisms based on previously reported effects in Caucasian patients in order to compare the genotype effects seen in each ethnicity. We anticipated that the increased prevalence of high-grade toxicities in Asians would yield increased statistical power in determining relevant genetic markers.A total of 97 mRCC patients who received sunitinib between 2006 and 2014 at the National Cancer Centre Singapore (NCCS) were included in this retrospective study. The study was approved by the Institutional Review Board  and written informed consent was obtained from each patient. Sample size estimation is detailed in Sunitinib toxicities and best radiological response were evaluated based on CTCAE version 3.0  and RECIThe toxicities assessed include leucopenia, neutropenia, thrombocytopenia, hepatotoxicity, diarrhea and hand-foot syndrome. Blood cell counts from the electronic medical system and physician notes from the first sunitinib cycle were assessed for leucopenia (<3000/μL), neutropenia (<2000/μL), thrombocytopenia (<150000/μL), hand-foot syndrome  and diarrhea (documented patient complaints). Hepatotoxicity was defined as elevation of AST (>33IU/L) or ALT (>36IU/L) above a normal baseline (AST≤33IU/L and ALT≤36IU/L) during the first two cycles.Progression-free survival (PFS) was defined as the time from the date of sunitinib initiation to the date of sunitinib termination when sunitinib was terminated due to radiological or clinical evidence of progressive disease (PD), severe toxicities or death, with termination due to PD and death due to PD as events. Dose reduction did not count as an endpoint for PFS. Overall survival (OS) was defined as the time from the date of sunitinib initiation to the date of death or to the date of the last follow-up for censored cases.FLT3 738 T/C, VEGFR2 1191C/T, ABCG2 421C/A, ABCB1 3435C/T, ABCB1 1236T/C, ABCB1 2677G/TA, as well as an intron 2 deletion polymorphism of BIM [BIM deletion are provided in We genotyped 6 SNPs in 4 genes, including m of BIM . The SNPGermline DNA was obtained from the buffy coat or from formalin-fixed tissue of benign kidney obtained from nephrectomy. The labeling on blood tubes and tissue slides were de-identified by a third party before they were used for DNA extraction. Genotyping was done by PCR amplification of the flanking region of each SNP followed by direct sequencing.P<0.20 were further analyzed using multivariate logistic regression including patient age, gender, baseline ECOG status and starting dose as covariates. PFS and OS were estimated using the Kaplan-Meier method [P<0.20 were further analyzed using a multivariate Cox regression model by including patient characteristics which had univariate P values of less than 0.05 as covariates and PFS or OS as the depending variable. Only patients for whom sunitinib was the first line treatment for mRCC were included in PFS and OS analyses. In all analyses, missing data were kept missing except for baseline ECOG status, which was replaced with the median value. With an exploratory purpose, multiple testing correction was not done.Genotype associations with toxicity events or best radiological response were first analyzed using univariate logistic regression. Genotypes generating r method . Univarir method  or a CoxBIM deletion are listed in P>0.05) [ABCB1 3435C/T, ABCB1 1236C/T and ABCB1 2677G/TA using a Chi-square test (P< 0.05 in each pair) and phasing with PLINK [TTT was found to be the most common haplotype. It was found in 51 patients, among whom 8 were homozygous carriers. A complete list of haplotypes and their frequencies is provided in The demographic and baseline clinical characteristics of the 97 patients included in this study are listed in  P>0.05) . After vth PLINK , haplotyFLT3 738 TT genotype was associated with an 8.0-fold increase in the risk of leucopenia (P = 0.03) and a 2.7-fold increase in the risk of neutropenia (P = 0.04). The ABCB1 1236 T allele, ABCB1 3435 T allele, ABCB1 2677 T allele, ABCB1 3435, 1236, 2677 TTT haplotype and the ABCG2 421 A allele were correlated with a 3-fold (P = 0.03), 10-fold (P = 0.01), 3-fold (P = 0.04), 10-fold (P = 0.03) and 3-fold (P = 0.03) decrease in the risk of neutropenia respectively. The ABCB1 1236 T and ABCB1 3435 T alleles were correlated with a 25-fold (P = 0.0005) and 3-fold (P = 0.02) decrease in the risk of diarrhea respectively. No genotypes were correlated with thrombocytopenia, hepatotoxicity or hand-foot syndrome. The VEGFR2 1191C/T genotype and BIM deletion were not associated with the toxicity endpoints.Univariate and multivariate logistic regression analyses for associations between genetic markers and clinical outcomes are listed in ABCB1 3435 TT genotype (P = 0.02), ABCB1 2677 TT genotype (P = 0.01) and the ABCB1 3435, 1236, 2677 TTT haplotype (P = 0.004)  and OS (P = 0.005)  . Median = 0.004) , after iFLT3 738 TT genotype predisposed to sunitinib-related leucopenia, an association which had previously been previously reported by van Erp et al. in Caucasian patients [CYP1A1 2455A/G  noted by van Erp et al. [et al. also correlated Asian ethnicity with a 13% decrease in sunitinib clearance and 15% increase in peak serum sunitinib concentration and area under curve compared to a control group that was composed of >85% Caucasians [We observed that the patients . The effpatients . This map et al.  but not ucasians . The incABCB1 2677T allele was associated with reduced neutropenia risk and inferior radiological response and the ABCB1 1236 T allele was associated with reduced risk of neutropenia and diarrhea. A trend was observed for the association of the ABCB1 1236 T allele with inferior PFS (P = 0.09) and OS (P = 0.07), which was previously reported in Caucasians [et al., whose study correlated ABCB1 1236 TT and ABCB1 2677 TT to increased clearance of sunitinib and its active metabolite in 114 cancer patients using univariate analyses that did not include demographic covariates [et al.’s findings that mRCC patients who received sunitinib as first-line therapy and carried ABCB1 1236 TT or ABCB1 2677 TT/TA require fewer dose reductions due to toxicities compared to carriers of other genotypes [et al. noted that the effect size of a single genetic polymorphism on clearance is much smaller than that of inter-individual variability and is thus inadequate to directly guide dosing [The ucasians . This asvariates . It is aenotypes . One plae dosing . TherefoABCG2 421 AA genotype was associated with reduced risk of neutropenia  appearset al. [The limitations of this study include the retrospective nature of our data collection and the attenuated dosing regimens adopted in Singapore to reduce toxicity. Indeed, we observed lower toxicity incidences as compared to that of the recent COMPARZ trial . For exaet al.  Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.


Microsatellites are DNA sequences consisting of repeated, short (1–6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level. Microsatellite DNA, also known as short tandem repeats (STR) or simple sequence repeats, are tracts of mono- to hexanucleotide repeats of variable length that are widely distributed in the genomes of animals, plants and microbes . MicrosaDue to their high degree of allelic variability, microsatellites are popular markers in population genetics , linkageSMRT sequencing , as implHere, we evaluate the potential of PacBio SMRT sequencing for highly multiplexed microsatellite length scoring. This technology offers moderate output relative to other NGS platforms, but at a considerably lower cost, making it appropriate for projects where the number of required sequence reads run only into the tens of thousands, but that may require longer reads that that produced by e.g. the Illumina platform. Essentially a microsatellite length scoring protocol using SMRT sequencing would be analogous to regular amplicon sequencing. The marker of interest is amplified by PCR using primers containing an additional short index sequence specific for the sample being amplified. PCR products are then quantified before pooling all of the samples, each containing a unique index sequence. The pool is cleaned before use as input for SMRTbell library construction per the manufacturer’s instruction, followed by the actual sequencing step. The output sub-reads are then subject to further analyses in order to generate CCS and carry out quality filtering. The resulting sequence file is demultiplexed using the list of unique sample indices, before length scoring and other biological analysis.In vitro experiments with Taq polymerase have shown that slippage rates increase with allele length while decreasing with repeat unit length, and that slippage is biased toward microsatellite contraction [When performing typical microsatellite length scoring analysis, artificially introduced polymorphisms are commonly introduced as a result of PCR amplification prior to scoring . The rattraction . Here, wWe designed two DNA constructs, each containing two microsatellites. Construct 1  is compo2O, incubation at 37°C for 4 hours). Digested fragments were separated by electrophoresis on a 1% agarose gel. Constructs were isolated using the QIAquick gel extraction kit according to instructions supplied by the manufacturer. Plasmid pBR329 [2O), producing plasmids pBR329-C1 (with construct 1) and pBR329-C2 (with construct 2) carrying disrupted resistance markers for ampicillin and chloramphenicol, respectively  PstI or EcoRI enzyme and 21 μl Hd pBR329  . PCR product was evaluated by 1% agarose gel electrophoresis. Successful MG1655 transformants with either pBR329-C1 or pBR329-C2 were stored at -80°C in 20% glycerol LB. These frozen cell stocks were used for all subsequent cultures for plasmid extractions.Transformation was verified in two ways. First, positive isolates were grown overnight in 10 μg/ml tetracycline LB medium at 37°C followed by plasmid extraction using the QIAGEN Plasmid Mini Kit according to instructions provided by the manufacturer. Plasmids were digested with either PstI (pBR329-C1) or EcoRI (pBR329-C2) as described above, using 5 μl of plasmid extract. Presence of the inserts was evaluated by visualization of appropriate size bands on a 1% agarose electrophoresis gel. Second, insert presence was verified by dissolving positive colonies in 25 μl of mqHt flanks  . Forward primers were appended 8 bp barcodes at the 5' end to allow for multiplexed sequencing  followed by extraction of pBR329-C1 and pBR329-C2 using the QIAGEN Plasmid Midi Kit according to the manufacturer's manual. Plasmid extractions were confirmed by 1% agarose gel electrophoresis and quantified using a NanoDrop ND-1000 spectrophotometer (v. 3.3). PCR was conducted using primer pairs specific for the constructs’ flanks  in 50 μlquencing . ReactioIn order to determine if the PCR reactions were saturated at high cycle numbers we ran three additional reactions for each cycle regime for each construct, using the same DNA stocks as for the sequencing experiment. Yields were then quantified with a Life Technologies Qbit fluorometer (model 1.0).2O. Plasmid purification was confirmed by running 5 μl of DNA on a 1% agarose gel. Constructs were excised from plasmids by EcoRI and PstI  digestion enzymes by adding 5 μl x10 NEB3 buffer and 0.5 μl of appropriate enzyme and digesting overnight at 37°C. Plasmid backbones and inserts were separated on a 1% agarose gel, and fragments were visualized using a Safe Imager Transilluminator (Invitrogen). Inserts were subsequently excised and purified using a QIAquick gel extraction kit.In order to distinguish between errors introduced by SMRT sequencing and PCR, no-PCR controls were created by excising constructs from pBR329-C1 and pBR329-C2 by PstI and EcoRI enzyme digestion, respectively. Plasmid-carrying strains were grown overnight from the same frozen stocks at 37°C in 50 ml selective LB media (100 μg/ml Ampicillin + 10 μg/ml Tetracycline or 10 μg/ml Chloramphenicol and 10 μg/ml Tetracycline), i.e. the exact same conditions as above. Plasmids were extracted from cultures using the QIAGEN Plasmid Midi Kit and re-suspended in 50 μl mqH2O to equal concentrations. The four pools were then combined in equal volumes for a total of 222 ng PCR amplicons and 13.9 ng of each no-PCR control for a total of 249.8 ng of DNA. The resulting pool was purified using the Pacific Biosciences AMPure PB prep kit according to instructions supplied by the manufacturer, and the final SMRT sequencing library was prepared using the Pacific Biosciences 500bp library preparation protocol. Fragment size was confirmed on an Agilent 2100 Bioanalyzer, and the library was sequenced on a PacBio RS II instrument using P4-C2 chemistry on one SMRT cell, with a movie time of 180 minutes. Quality filtering was performed using the “Reads Of Insert” protocol on SMRT portal . Filtering was done with the minimum number of passes set to 3 for circular consensus sequences (CCS) (The four PCR reactions from each of the four cycle number regimes were pooled and cleaned using a QIAquick PCR Purification Kit. Each pool was quantified using a Life Technologies Qbit fluorometer (model 1.0) according to standard manufacturer's protocol and diluted with mqHes (CCS)  and a mies (CCS) –S4 Fileses (CCS) . All staThe average distributions of slippage errors by integer numbers of repeat units, for all three microsatellite structures, are presented in We first assessed the effects of PCR relative to the no-PCR controls by comparing the absolute errors of control reads to reads resulting from different PCR regimes , includiFor each PCR regime we calculated the percentage of non-target length sequence reads that corresponded with either deletions or insertions by integer numbers of repeat units . CompariWe calculated the percentage of non-target length sequence reads that did not correspond with deletions or insertions by integer numbers of repeat units . These eFor comparison with non-microsatellite DNA we looked at indel patterns in the linker sequences separating the two microsatellites on each construct. These DNA sequences are essentially random and thus suitable for obtaining baseline indel rates introduced through PCR and SMRT sequencing. For these sequences we also observed a large predominance of insertion to deletion events Click here for additional data file.S2 FigConstruct 1 disrupts ampR gene, allowing for selection for ampicillin-sensitive hosts. Construct 2 disrupts camR gene, allowing for selection for chlorampenicol-sensitive hosts. Both vectors provide tetracycline resistance to host.(PNG)Click here for additional data file.S3 FigThe total number of reads for each experimental treatment in indicated on the y-axis. Experimental treatment is indicated is indicated on the x-axis. EcoRI and pstI are the no-PCR controls. Construct number (C1 or C2) is indicated first in the other x-axis labels, followed by replicate number (1 or 2), followed by number of PCR cycles (10–40).(BMP)Click here for additional data file.S4 FigThe y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S5 FigRepeat length distribution for 30xCA sequence, 10 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S6 FigRepeat length distribution for 30xCA sequence, 20 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S7 FigRepeat length distribution for 30xCA sequence, 30 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S8 FigRepeat length distribution for 30xCA sequence, 40 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S9 FigThe y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S10 FigRepeat length distribution for 20xATCT sequence, 10 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S11 FigRepeat length distribution for 20xATCT sequence, 20 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S12 FigRepeat length distribution for 20xATCT sequence, 30 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S13 FigRepeat length distribution for 20xATCT sequence, 40 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S14 FigThe y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S15 FigRepeat length distribution for 10xATCT sequence in construct 1, 10 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S16 FigRepeat length distribution for 10xATCT sequence in construct 1, 20 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S17 FigRepeat length distribution for 10xATCT sequence in construct 1, 30 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S18 FigRepeat length distribution for 10xATCT sequence in construct 1, 40 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S19 FigThe y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S20 FigRepeat length distribution for 10xATCT sequence in construct 2, 10 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S21 FigRepeat length distribution for 10xATCT sequence in construct 2, 20 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S22 FigRepeat length distribution for 10xATCT sequence in construct 2, 30 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S23 FigRepeat length distribution for 10xATCT sequence in construct 2, 40 PCR cycles replicates 1 (A) and 2 (B). The y-axis indicates the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axis.(BMP)Click here for additional data file.S24 FigEach point in the plots represents an absolute error of the magnitude (in bp) indicated on the y-axes, while the number of PCR cycles is indicated on the x-axes. Each band in the figure illustrates the density of reads deviating from the target length (zero on y-axes), with a small amount of jitter, both horizontal and vertical, added to the plotted values for enhanced visualization and interpretability. For the same reason extreme outliers have been omitted from the plots. The red lines are mean absolute percentage errors (including outliers). For each PCR regime all experimental replicates have been combined. A. 30xCA repeat sequence. B. 20xATCT repeat sequence. C. 10xATCT repeat sequence.(BMP)Click here for additional data file.S25 FigThe lines are fitted GAMs with 2 s.e. confidence regions delimited by the shaded bands. Panels A-C include the restriction digest controls while D-F do not. A. 30xCA: p = 0.058. B. 20xATCT: p<0.001. C. 10xATCT: p<0.001. D. 30xCA: p = 0.479. B. 20xATCT: p = 0.028. C. 10xATCT: p = 0.123.(BMP)Click here for additional data file.S26 FigThe lines are fitted GAMs with 2 s.e. confidence regions delimited by the shaded bands. Panels A-C include the restriction digest controls while D-F do not. A. 30xCA: p = 0.059. B. 20xATCT: p = 0.002. C. 10xATCT: p<0.001. D. 30xCA: p = 0.373. B. 20xATCT: p = 0.400. C. 10xATCT: p = 0.185(BMP)Click here for additional data file.S27 FigYield from PCR reactions using 10, 20, 30 and 40 reaction cycles for constructs 1 (A) and 2 (B). Each reaction was done in triplicate. Error bars are ± 1 s.e.(BMP)Click here for additional data file.S28 FigRelationship between stepwise deletion (A-C) and insertion (D-F) mutations and PCR cycle number. The y-axes indicate the percentage of sequence reads corresponding to either stepwise deletions or insertions. A and D: 30xCA. B and E: 20xATCT. C and F: 10xATCT. All models (GAMs) are statistically significant. A. 30xCA: p = 0.004. B. 20xATCT: p<0.001. C. 10xATCT: p = 0.001. D. 30xCA: p = 0.005. E. 20xATCT: p = 0.024. F. 10xATCT: p = 0.006.(BMP)Click here for additional data file.S29 FigRelationship beween stepwise deletion (A-C) and insertion (D-F) mutations and PCR cycle number, omitting the no-PCR controls. The y-axes indicate the percentage of sequence reads corresponding to either stepwise deletions or insertions. A and D: 30xCA. B and E: 20xATCT. C and F: 10xATCT. Only the models (GAMs) shown in A and D are statistically significant .(BMP)Click here for additional data file.S30 FigAll pairwise differences are statistically significant: 30xCA: p = 0.014, 20xATCT: p = 0.004, 10xATCT: p<0.001, linker: p<0.001 (paired Wilcoxon signed rank test). I–insertion. D–deletion.(BMP)Click here for additional data file.S31 FigA-C: insertions. D-F: deletions. None of the relationships are statistically significant . A. 30xCA: p = 0.341. B. 20xATCT: p = 0.771. C. 10xATCT: p = 0.949. D. 30xCA: p = 0.628. E. 20xATCT: p = 0.708. F. 10xATCT: p = 0.071.(BMP)Click here for additional data file.S32 FigThe y-axis shows the percentage of total sequence reads with an indel mutation in the spacer region of the constructs. The trend line is from a linear regression model (p = 0.006). GAM modelling also indicated a linear fit.(BMP)Click here for additional data file.S33 FigSequence type and indel direction (I–insertions. D–deletion) are indicated on the x-axis(BMP)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S2 FileUtility functions for carrying out analysis.(R)Click here for additional data file.S3 FileRead and structure data.(R)Click here for additional data file.S4 File(R)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableAll barcodes were added to 5'-end of forward primers.(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Traditional genotyping assays detect viral variants present in at least 15–25% of the entire virus population. We tested the Next generation GS Junior System (NGS) setted with a detection limit of 0.05% and evaluated the clinical relevance of low prevalent mutations.NGS was performed on the plasma of 26 infected individuals who started a TDF/FTC/RPV (15 subjects) or TDF/FTC/EFV (11 subjects) cART after a routine HIV-1 drug-resistance negative test by Viroseq HIV-1 Genotyping System. Amplicon Sequencing of HIV-1 RT and PR Plate (Roche) was performed following the manufacturer's instructions. HIV-1 variants were analyzed by a specific HIV-1 tool by AVA software v. 2.7. The updated IAS resistance mutations list (March 2013) was considered for the analysis of resistance positions. Patients were followed testing viral load and immunologic parameters.Twenty four males and two females with a mean age of 43 years were included. Twenty-one were nave for cART. At baseline, median HIV-RNA was 4.57 log copies/mL (range 2.15–6.57) and CD4 count 315 cells/mcL (range 16–648). In 18 patients, NGS did not detect any additional variant relevant for the selected cART compared to population genotyping. In the remaining eight patients resistance conferring mutations to part of the ongoing regimen were detected. Single mutations E138K (two cases) and M184V in three distinct patients and V90I+G190E; M184V+A98S; Y215F+V118I+T215I; L210S+T215I+F227L; and A62V+D67G+K70N+188H in the remaining five subjects. In all cases, the mutation prevalence was inferior to 5%. The mean daily reduction of VL was −3759 copies/mL in patients without NGS detected mutations and −1045 copies/mL in those with mutations. The median KM estimates for reaching an HIV-RNA blood level <50 copies/mL were 127 days and 161 days, respectively. One patient without baseline resistance selected for M184I+E138K+T215I (NGS) after four months of TDF/FTC/RPV therapy.NGS detected low-frequency HIV-1 variants harbouring RT drug resistance mutations that could have affected the therapy outcome. However, viral decay in an early cART phase was not affected by the presence of resistant minority variants. The low prevalence of the detected mutation, the limited effect on the combination regimen and the potency of cART components could be possible explanations of our findings. Longer follow-up and larger casuistries are needed to determine the clinical relevance of NGS in routine clinical practice and eventually define a clinically relevant mutations' prevalence.


C of BoNT/A. Here, we deciphered the BoNT/A‐SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C >> SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A‐SV2C interaction is of different nature compared to BoNT/B‐Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone–backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A‐SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic.Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A‐C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain H Clostridium botulinum are the most poisonous protein toxins known [N , HCN and HCC (25 kDa each). The latter two domains constitute the HC-fragment and are responsible for neuronal binding [Botulinum neurotoxins (BoNTs) produced by the bacteria ns known . They cans known . The sevns known ,4. BoNTs2+-metalloprotease, is translocated through the pore formed by the HN domain into the cytosol of the cell and is released after reduction of the disulfide bridge connecting LC and HC [N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein takes place; in the case of BoNT/A, synaptosome-associated protein of 25 kDa (SNAP-25) is specifically cleaved at Q197-R198 [Here, the initial low affinity binding to complex polysialo-gangliosides and subsequent high affinity binding to different synaptic vesicle protein receptor takes place . After tC and HC . Here, tBoNT/A, B, E, F and G all possess a single ganglioside binding site (GBS) comprising the conserved E(Q)…H(K)…SxWY…G motif ,10,11, wC [CN and HCC [CC domains in close proximity to the single GBS [The three identified highly homologous isoforms SV2A, B and C comprise twelve putative, conserved transmembrane domains TMD; A, are loC ,23. The C . The cor and HCC ,30 whilengle GBS ,31,32,33ngle GBS ,30. The ngle GBS , which, ngle GBS . The homEscherichia coli (E. coli). GST pull down experiments showed that BoNT/A binds preferable to rSV2C-LD4 lacking the C-terminal TMD8, whereas addition of complex polysialo-gangliosides does not alter the interaction. HCA displays the highest binding affinity to hSV2C and rSV2C, but much weaker affinity to rSV2A and B. Accordingly, SV2-LD4 peptides fused to the BoNT/A C-terminus drastically reduced the potency of BoNT/A at the neuromuscular junction. A KD of 200 nM was determined for full-length BoNT/A binding to GST-rSV2C-LD4 in pull down experiments which agrees well with the KD of 550 nM determined for HCA to GST-rSV2C-LD4 by surface plasmon resonance (SPR) measurements. The F563 in hSV2C-LD4 significantly enhanced BoNT/A binding to hSV2C-LD4 vs. rSV2C-LD4 displaying a leucine instead. Circular dichroism (CD) spectroscopy revealed that the quadrilateral helix of SV2C-LD4 pre-exists in solution prior to BoNT/A binding. In comparison to the BoNT/B–Syt interaction, the recognition of SV2 by BoNT/A follows a different mechanism. In summary, these data further deepen the understanding of the BoNT/A-SV2 receptor interaction and provide a basis for developing peptidic BoNT/A inhibitors as botulism countermeasures or high affinity peptides for implementation in innovative detection systems to capture functional BoNT/A variants, e.g., from complex matrices.In this study, we further characterized the protein–protein interaction of BoNT/A with its protein receptors SV2A-C. The LD4 of rSV2A-C and hSV2C were expressed as glutathione-S-transferase (GST) fusion proteins and hSV2C was also expressed as His-tagged fusion protein in in vitro that the presence of gangliosides synergistically increases the affinity to Syt-I and Syt-II incorporated into detergent micelles via their TMD. However, in the absence of gangliosides the affinity of Syt-I and Syt-II with TMD to BoNT/B and G decreases below that of Syt-I and Syt-II without TMD, presumably due to steric constraints at the micelle surface [The neuronal uptake of BoNT/A, B, E and G occurs via a double receptor binding mechanism employing a complex polysialo-ganglioside as well as a synaptic vesicle protein receptor ,14,30,35 surface . Similar surface , presenc surface , ganglioCA  is only 47.6% and 44.0% identical to rSV2A 468–594 and rSV2B 411–535, respectively A. The laCA . HCA dis 454–579 , howeverD of full-length BoNT/A to rSV2C has not yet been determined. For that purpose, we performed a pull down assay with immobilized GST-rSV2C 454–579 (75 pmol) and increasing concentrations of single chain H6tBoNTA (31 nM–1 µM). Fitting a function to the saturation binding curve as well as a Scatchard plot analysis revealed a KD ≈ 200 nM for H6tBoNTA  than to rSV2C LD4. As indicated in To verify whether the binding of full-length BoNT/A to hSV2C differs compared to rSV2C, tag-free fully activated BoNT/A  was test2+ or Co2+ to the N- and C-termini of hSV2C-LD4, respectively, yielding H6hSV2C6xHN and as a control hSV2C6xHN comprising only the C-terminal 6xHN-tag. Interestingly, placement of the small affinity tag to the C-terminus mimicking the presentation of the LD4 via its TMD8 increased binding of BoNT/A to hSV2C6xHN compared to GST-hSV2C comprising a much larger N-terminal tag and a freely floating SV2C C-terminus. However, addition of the H6-tag to the N-terminus and hence presenting SV2C-LD4 fixed on both termini like in full-length SV2C on the SV surface did not alter the binding to BoNT/A . Lowering the pH to values being measured in acidifying small synaptic vesicles [KD increases at least an order of magnitude. The lowered affinity of HCA towards the F563L mutant observed in the pull down assay could be confirmed by the SPR measurements. The affinity of HCA towards the GST-hSV2C F563L mutant was statistically significantly  reduced by a factor of 1.8 as compared to GST-hSV2C wild-type. However, the effect was not as pronounced as in the pull down assay although one has to keep in mind that for SPR measurements HCA instead of full-length BoNT/A was used.Overall, the observed interactions were characterized by rapid binding and fast dissociation and calculated n assays , hSV2C svesicles  the KD iC-terminus of BoNT/A to block the SV2 binding site. The BoNT/A–SV2 fusion proteins of up to 165 kDa were successfully generated  MtMfpA [Cyanothece species 51142 [CA yields a spectrum of a protein comprising mainly β-sheets and is similar to spectra previously determined for HCB and HCG [CAS and hSV2C peptide were mixed in a 1:1 stoichiometry. The immediate measurement yielded a spectrum dominated by β-sheet content  indicating that no alteration in secondary structure content occurred in HCA and hSV2C, respectively. Conversely, the CD signal of free hSV2C or HCAS was subtracted from the spectrum of the HCAS-hSV2C mixture. In both cases, the resulting difference spectrum was highly similar to that measured of the respective isolated protein. In conclusion, hSV2C seems to fold already in solution as quadrilateral helix and binding to HCA does not induce a change in its secondary structure.Isolated hSV2C displays a spectrum rich in β-sheet  MtMfpA ,40 or lues 51142 ,42,43. Ses 51142  for the es 51142 . AdditioThe recognition of SV proteins as receptors by botulinum neurotoxins is a highly critical step in their intoxication pathway. Following the low affinity accumulation of BoNT by the abundant complex polysialo-gangliosides on the neuronal surface only a high affinity interaction with the SV protein provides sufficient specificity and avidity to enable efficient uptake of the BoNT into the lumen of small SV.in vitro. Although we experimentally proved the double receptor recognition of BoNT/A recently [C-terminus important for micelle/membrane integration might fold back and binds to hydrophobic patches on the quadrilateral helix thereby blocking the surface used by BoNT/A. Alternatively, the TMD8 fails to orient the LD4 as required for BoNT/A to simultaneously bind SV2C and gangliosides. Obviously, the steric requirements for full-length BoNT/A to approach membrane juxtaposed SV2 LD4 are by far more demanding than for HCA which might explain the observation that isolated HCA displays increased binding to GST-rSV2C 454–603 in the presence of gangliosides compared to GST-rSV2C 454–579 lacking TMD8 [Using a similar experimental setup as previously used for BoNT/B–Syt-II  we analyrecently , we obseing TMD8 . Hence, CA 5–7-fold less than rSV2C, but determination of a binding constant KD by pull down as well as SPR assays for the interaction of BoNT/A and HCA vs. SV2A and SV2B, respectively, failed because of too low affinity. A similar qualitative order of binding affinity was previously observed [CA complex, ten side chain residues mediate sequence specific interactions. Of these ten residues eight are dissimilar in rSV2A and nine in rSV2B while four of them also differ between rSV2A and rSV2B. Such diversity partially explains the low affinity binding observed for BoNT/A to SV2A and SV2B and the absence of BoNT/E binding to SV2C. Analysis of the phenylalanine pentapeptide repeats causing the quadrilateral helix folding of LD4 revealed two sites each in SV2A and SV2B with a mispositioned or even lacking phenylalanine, which might impair stability or correct folding of the quadrilateral helix. Repositioning and/or replenishing the lacking phenylalanine led to increased protein solubility of the SV2A and SV2B mutants as well as increased their binding to BoNT/A [The SV2A-C isoforms display a high homology and share ~60% AA sequence identity, but only 44 of 125 residues in LD4 are identical in all three isoforms. One can expect that this moderate homology in LD4 will influence physiological function and more importantly, the recognition by BoNT/A and E. Indeed, BoNT/E is only able to interact with SV2A and SV2B but not SV2C ,35 whichobserved . Despiteo BoNT/A . Hence, vs. rSV2C must be ascribed to the four residues in LD4 interacting with BoNT/A, but differing between human and rat SV2C. Of the two residues with non-conservative substitutions , F563 of hSV2C has been reported to mediate an important cation–π-stacking interaction with R1156 of BoNT/A [CA, respectively. Interestingly, aromatic residues at position 563 are only present in human, chimpanzee, chicken, dog, zebrafish and pufferfish SV2C, but a correlation with BoNT/A potency in those species comprising a non-aromatic AA in SV2C has not been exhibited yet.The differences in affinity observed for hSV2C f BoNT/A , which cC-terminus of BoNT/A decreased its potency even 100-fold. This 10-fold higher effect may partially be attributed to the different location of Syt and SV2 binding sites in HCB and HCA, respectively. The SV2 site comprises inter alia the last five BoNT/A residues and fusion of any larger peptide might disturb SV2 binding. However, insertion of a 11-mer linker between the C-terminus of BoNT/A and the SV2C provides more flexibility of the latter to orient into its binding site and resulted in an additional 10-fold reduction of potency. Accordingly, N-terminal truncation of the rSV2C and replacement by rSV2A reversed potency indicating specific intramolecular inhibition of BoNT/A by SV2 peptides according to their binding site affinity.Complementary to the affinity determined by pull down assays, an intramolecular inhibition of protein receptor binding was conducted to evaluate the binding strength of SV2 peptides to BoNT/A. Previously, a BoNT/B–Syt-II fusion protein displayed >10-fold reduction in potency due to autoinhibition . Fusing CB–rSyt-II interaction displayed a fast association rate ka despite preceding folding of the Syt-LD α-helix and an intermediate dissociation rate kd yielding a binding constant of 75 nM which agrees well with the KD of 34 nM [CA binds to hSV2C as quick as HCB to Syt-II, but its dissociation rate is 3-fold faster presumably due to the weaker backbone–backbone interactions. Therefore, binding constants of 500 nM for GST-rSV2C-HCA in SPR measurements and 200 nM for GST-rSV2C-BoNT/A in pull down assay were calculated. Hence different methods using different ligands like HCA or full-length BoNT/A (which could not be measured in SPR for safety reasons) largely arrive at the same overall binding affinity. The slightly lower KD of ~400 nM for the interaction between GST-hSV2C 454–579 wild-type and HCA in SPR is in good agreement with previous affinity data of 260 nM determined for hSV2C 456–574 peptide by fluorescence anisotropy experiments [CA towards a hSV2C 529–579 peptide measured in another work [ka = 2.3 × 103 M−1s−1) and the dissociation rate constant (kd = 2.5 × 10−4 s−1) were significantly slower by a factor of 135 and 480, respectively. Jacky et al. [CA by amine coupling to a CM5 sensor chip yielding a random presentation. In contrast, we captured the GST-SV2C peptides by anti-GST antibodies, where the SV2C is uniformly oriented to the solvent. Furthermore, Jacky et al. [CA in physiological buffer over the sensor surface, hereby mirroring the natural presentation of the SV2C receptor on the surface of the presynaptic membrane [CA and denaturation of hSV2C by solvent [et al. [CA-hSV2C 529–579 interaction by the BIA simulation software 2.1 employing the 1:1 kinetic-binding model (A + B ↔ AB), the above cited ka, kd and KD and five hSV2C peptide concentrations surprisingly yields curves , single chain full-length BoNT/A equipped with an N-terminal, thrombin-cleavable His6tag (H6tBoNTA), full-length BoNT/A with thrombin-cleavable tags (BoNTA) as well as GST fusion proteins of rSV2A 468–594, rSV2B 413–535, rSV2C 454–579 and rSV2C 454–603 were described previously [Plasmids encoding the Heviously ,30,36.E. coli codon usage optimized DNA sequence encoding hSV2C 455–579  into a pGEX-4T3 vector cut with BamH I/Xma I. The mutation F563L was inserted into pGEXhSV2C 455–579 by PCR applying the GeneTailor™ site-directed mutagenesis system  and suitable primers . pGEXhSV2C-6xHN 455–579 was generated by inserting a DNA fragment encoding a 6xHN tag into pGEXhSV2C 455–579 via Xma I/Xho I sites. The plasmid encoding pGEXH6hSV2C-6xHN 455–579 was generated from pGEXhSV2C-6xHN 455–579 by inserting a DNA-fragment encoding a H6-tag employing the 5′ BamH I site.The plasmid pGEXhSV2C 455–579 encoding GST-hSV2C 455–579 was generated by cloning the synthetic Expression vectors encoding variants of BoNTA-SV2-S fusion proteins were generated by opening pBoNTAS wt at the bont/A 3′ end using the unique Sma I site  and inseNucleotide sequences of all newly generated constructs were verified by DNA sequencing .CAS, scBoNTAS, single-chain H6tBoNTA and activated full-length BoNTA were described previously [The generation of Heviously ,30,36. Eeviously .2 to cleave off the SV2C peptide from the GST-tag. The released (H6)hSV2C6xHN-peptides were isolated from the supernatant using Co2+-Talon matrix  and eluted with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 250 mM imidazole. The released hSV2C-peptide was purified from the supernatant by gelfiltration in PBS, pH 7.4. Desired fractions were pooled, dialyzed against PBS pH 7.4, frozen in liquid nitrogen and kept at −70 °C.GST fusion proteins , the AIDA 3.51 software  and BSA (100–1600 ng) as reference protein.The GST pull down assays were similarly performed as previously described  with the2+-Talon matrix , respectively, and subsequently incubated for 2 h at 4 °C with HCAS, single chain H6tBoNTA or di-chain BoNTA in a total volume of 100–200 µL in binding buffer as stated in the respective figure legends. Beads were collected by centrifugation and washed two times each with the corresponding binding buffer. Washed pellet fractions were incubated at 37 °C for 20 min in SDS sample buffer and analyzed by 10% SDS-PAGE. Protein bands were detected by Coomassie blue staining and subsequently quantified by densitometry using the software TINA . Unspecific binding of ligand to immobilized GST or free Co2+-Talon matrix was subtracted from the specific binding signal of HCA or BoNTA to SV2. KD values and statistics were calculated with GraphPadPrism .Briefly, GST, GST fusion proteins and 6xHN-tagged fusion proteins (75 or 150 pmol each) were immobilized to 10 µL glutathione-sepharose-4B matrix  or 5 µL CoCA-fragments towards GST-hSV2C 455–579 and GST-rSV2C 454–579 were performed using a Biacore X100 unit  at 25 °C. PBS (pH 7.3) or 51.4 mM Na2HPO4, 24.3 mM Citrate, 150 mM NaCl (pH 5.0) supplemented with 0.5% Triton X-100 were used as running buffer. For GST-rSyt-II 1–61, HBS-EP+  was used as running buffer. Recombinant GST-fusion proteins were immobilized employing the GST capture kit  according to the manufacturer’s recommendations. Briefly a CM5 sensor chip was covalently coupled with rabbit anti-GST capture antibodies. For each measurement, GST-hSV2C 455–579 (wt or F563L) or GST-rSV2C 454–579 and, as a negative control, recombinant GST (both at 5 µg/mL) were captured for 30 s at a flow rate of 5 µL/min on flow cells 2 or 1, respectively, leading to immobilization levels of approximately 200 resonance units (RUs).SPR measurements to determine binding kinetics and/or affinity of recombinant HCA or HcB  were injected over both flow cells for 60 or 120 s at a flow rate of 30 µL/min. Dissociation was monitored subsequently for 120 or 300 s by running buffer injections. Between measurements, the sensor surface was regenerated by injections of 10 mM glycine-HCl (pH 2.1) for 120 s over both flow cells. Due to incomplete regeneration for GST-rSV2C 454–579 measurements and GST-hSV2C 455–579 (pH 5.0), an additional injection of 10 mM NaOH for 15 s at 10 µL/min was necessary to completely remove bound HCA from the flow cells. Duplicate injections of the highest HCA concentration as well as triplicate injections of running buffer only were included. Due to the harsh regeneration conditions necessary during GST-rSV2C 454–579 and pH 5.0 measurements, a gradual loss of immobilization levels was observed. Therefore, the duplicate measurement of the highest HCA concentration had to be omitted from this analysis to enable robust global fitting of binding affinities.For kinetic measurements, three-fold dilution series of Hka and kd) and the equilibrium dissociation constant KD were determined by fitting double referenced sensorgrams [max set global using the Biacore evaluation software (v2.01). KD values were additionally determined by fitting to a steady-state affinity model as a test for consistency between kinetic and affinity analysis of the interactions.Kinetic association and dissociation binding rate constants . Isometric contractions (resting tension ~10 mN) were recorded with a force transducer  and the software VitroDat , Seeheim, Germany). Toxin was applied in 4 mL of Earle’s Balanced Salt Solution supplemented with 0.1% BSA in concentrations to allow 50% decay of the contraction amplitude within 50 to 150 min. The times required to decrease the amplitude by 50%  for three BoNT concentrations were used to construct the calibration curves for scBoNTAS to which a power function was fitted  = 108.61x−0.355, R2 = 0.894). This power function was used to convert the paralysis times t½ determined for BoNTA-SV2 constructs into the corresponding scBoNTAS wt concentrations and expressed as relative biological activity of scBoNTAS wt.Intramolecular inhibition of BoNT/A by fusing various SV2 peptides to the PN assay ,38. In bCA and 5 µM hSV2C in PBS pH 7.4. Spectra were recorded at 22 °C from 195 to 250 nm with 100 nm/min, response of 1 s, standard sensitivity, bandwidth of 1 nm and five accumulations. Spectra were analyzed, processed and visualized using Spectra Manager II software .Circular dichroism (CD) data was collected with a Jasco J-810 spectropolarimeter  in a 1 mm path length cuvette with a concentration of 5–8 µM degassed H


Y358C mice revealed facilitatedphosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 andβ-Arrestin in hippocampus and amygdala. In behavioral assays,PDE4BY358C mice displayed decreased anxiety and increased exploration,as well as cognitive enhancement across several tests of learning and memory,consistent with synaptic changes including enhanced long-term potentiation andimpaired depotentiation ex vivo. PDE4BY358C mice alsodemonstrated enhanced neurogenesis. Contextual fear memory, though intact at24 h, was decreased at 7 days in PDE4BY358C mice, an effectreplicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMPsignaling by PDE4B in a very late phase of consolidation. No effect of thePDE4BY358C mutation was observed in the prepulse inhibition and forcedswim tests. Our data establish specific inhibition of PDE4B as a promisingtherapeutic approach for disorders of cognition and anxiety, and a putative targetfor pathological fear memory.Cognitive dysfunction is a core feature of dementia and a prominent feature inpsychiatric disease. As non-redundant regulators of intracellular cAMP gradients,phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant tolearning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is animportant phosphodiesterase in the hippocampal formation, is a major Disrupted inSchizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatricillness. To define the effects of specific inhibition of the PDE4B subtype, wegenerated mice with a catalytic domain mutant form of PDE4B (Y358C) that hasdecreased ability to hydrolyze cAMP. Structural modeling predictions of decreasedfunction and impaired binding with DISC1 were confirmed in cell assays. Phenotypiccharacterization of the PDE4B Cognitive dysfunction is a core feature of dementia and a prominent feature in majorpsychiatric disorders, such as mood and chronic psychotic disorders. Consequently,there is a large unmet need for cognition-enhancing drugs. The second messengercyclic adenosine monophosphate (cAMP) mediates fundamental aspects of brain functionrelevant to learning, memory, and higher cognitive functions (N-methyl-D-aspartate receptor antagonist MK-801 (The PDE4 family is cAMP-specific and comprises four subtypes (A–D). Theexpression patterns of individual PDE4 subtypes are clearly distinct at the regionaland cellular level, suggesting that PDE4 subtypes serve non-redundant functions.Non-subtype-selective brain-penetrant PDE4 inhibitors , such as rolipram, have shown therapeutic benefit in preclinical models ofpsychiatric and neurological diseases  and 2 (UCR2) . The cAMPde4b KOmice show an increase in the proliferation of neuronal cells in the hippocampaldentate gyrus  that has decreased ability to hydrolyzecAMP. The catalytic domain of PDEs is an important pharmacological target, givenlimited homology between subtypes , we detected an adenine to guanine (A1073G) transition,corresponding to a Tyr358 (TAC)→Cys (TGC) (Y358C) exchange stretches fromamino-acid residues (a.a.) 305–690 and is encoded by exchange. The exoorms1–5  and iscorms1–5 .2 backcross progeny of the founderPDE4BY358C/+ (C3H/HeH × BALB/cAnN)F1 male and wild-type (WT) C57BL/6NTac females were backcrossedthrough the male and female lines to C57BL/6J for 10 generations beforeheterozygotes were intercrossed to generate homozygous mutant(PDE4BY358C/Y358C) and WT (PDE4B+/+)littermates for phenotypic characterization. PDE4BY358C/+frozen embryos are available from the MRC Mammalian Genetics Unit, UK(har.mrc.ac.uk).Heterozygous NFull methods are available in the t-test, linear regression, repeated measures ANOVA, and Coxregression. Post hoc tests were performed using least significantdifference when significant genotype*test interactions emerged in ANOVA orrepeated measures ANOVA.Sex-differences were explored with two-way analysis of variance (ANOVA), however,no significant Genotype × Sex interactions were observed. For parsimoniousinterpretation, statistical differences are reported using Student’sAt the cAMP binding site, there is an interaction between the central phosphate groupof cAMP and H406 in WT PDE4B1 . Though Y358C/Y358C and PDE4B+/+ mice , which wC brains . WesternC brains , but was58Cmice . The DIS58Cmice . β-Arres58C mice , β-Arresamygdala ,but notamygdala .Y358C/Y358C mice spent moretime in the open arms and made more exploratory head dips and passages thanPDE4B+/+ mice  memory of a familiar juvenile compared withPDE4B+/+ mice  were administered to PDE4Bfreezing , supportY358C/Y358C mice. The Y358C mutation did notaffect basal synaptic transmission  trains and varied their number. Following tetanic stimulationwith four trains, PDE4BY358C/Y358C slices demonstrated enhancedpotentiation  or vehicle twice daily for 6 days. Increased numbers ofdentate BrdU+ and doublecortin+ cells were observed inPDE4BY358C/Y358C mice compared with both rolipram-treated andvehicle-treated PDE4B+/+ mice  that has decreased ability to hydrolyzecAMP. Consistent with previous data suggesting the involvement of the Y358 residue inthe interaction of PDE4B with DISC1  fearmemory at 24 h, which is considered sufficient time for the formation oflong-term memories  and the foot-shock(unconditioned stimulus) . The defY358C/Y358C phenotype, these previous studies detected nochange in fear memory at 7 days if the interventions occurred at or after 24 h, the United Kingdom Medical Research Council (G0900625)and the National Alliance for Research on Schizophrenia and Depression (to SJC). JCRholds a Canada Research Chair in Learning and Memory. The remaining authors declareno conflict of interests.


Vitamin D has been suggested to be active as an immunomodulator in autoimmune diseases such as Hashimoto’s thyroiditis (HT). The goal of the present study was to investigate the vitamin D status in HT patients.This prevalence case-control study was conducted on 90 patients with HT (of ages 12.32±2.87 years) and 79 age-matched healthy controls (11.85±2.28 years). Serum 25-hydroxyvitamin D3 [25(OH)D3] levels were measured in all 169 subjects.The prevalence of vitamin D deficiency in HT patients  was significantly higher than that in the control group  (p=0.025). Mean serum 25(OH)D3 level in the HT group was significantly lower compared to the control group . HT was observed 2.28 times more frequently in individuals with 25(OH)D3 levels <20 ng/mL .Vitamin D deficiency is associated with HT in children and adolescents. Levels lower than 20 ng/mL seem to be critical. The mechanism for this association is not clear. Vitamin D is a secosteroid hormone which is activated by enzymes and acts via receptors. In the liver, vitamin D is hydroxylated to 25-hydroxyvitamin D3 [25(OH)D3], which is the major circulating and stored form of the hormone and also the form that is measured to determine vitamin D status. A further hydroxylation step is required in the kidneys to convert 25(OH)D3 into the biologically active form of 1.25-dihydroxyvitamin D [1.25(OH)2D3] (1.25(OH)2D3 interacts with its vitamin D nuclear receptor which is present in the small intestine, kidneys and other tissues . In addiThere are various experimental models studying the immunomodulatory effects of 1.25(OH)2D3 and its analogues in autoimmune disease. It has been reported that in lpr/lpr mice systemic lupus erythematosus , experimIn this study, we aimed to determine if HT patients were vitamin D-deficient by comparing their serum vitamin D levels with those of healthy controls.The serum vitamin D levels of 90  newly diagnosed HT patients between ages 3.64 and 17.16 years (mean: 12.32±2.87) who had presented to the Cerrahpaşa Faculty of Medicine Pediatric Endocrinology Clinic were compared with those of an age-and sex-matched healthy control group (n=79) . The conNone of the patients had been treated with L-thyroxine (LT4) at the time the samples were taken. The diagnosis of HT was based on the following findings: anti thyroid peroxidase (anti-TPO) levels >35 IU/mL, anti-thyroglobulin (anti-Tg) levels >40 IU/mL and parenchymal heterogeneity on thyroid ultrasound. Patients with renal and liver disorders, diabetes mellitus, metabolic bone and parathyroid disorders or epilepsy treated by anticonvulsant therapy and patients on other medications that may alter 25(OH)D3 or 1.25(OH)2D3 metabolism and thyroid functions were excluded. The study was conducted between February 2013 and June 2014 and approved by the Cerrahpaşa Faculty of Medicine Ethics Committee . Written informed consent was taken from all parents and from children older than 16 years.Overt hypothyroidism (OHP) was diagnosed if serum thyroid stimulating hormone (TSH) was >5.7 µU/mL  and free T4 (fT4) was <0.7 ng/dL . Subclinical hypothyroidism was diagnosed if the serum TSH level was increased (>5.7 µU/mL), but serum fT4 and free triiodothyronine (fT3)  levels were within the reference ranges. Overt hyperthyroidism was diagnosed if serum TSH was <0.7 µU/mL and fT4 >1.9 ng/dL. Subclinical hyperthyroidism was diagnosed if serum TSH level was <0.7 µU/mL and serum fT4 and fT3 levels were within normal limits. The patients were considered to be euthyroid if their serum fT4 and TSH levels were within normal limits.Serum 25(OH)D3 levels ≤20 ng/mL, those between 21-29 ng/mL and ≥30 ng/ mL were accepted as vitamin D deficiency, insufficiency and sufficiency, respectively (21). Thus, patients and controls were grouped as 25(OH)D3 deficient, insufficient and sufficient. Patients with 25(OH)D3 deficiency, insufficiency and sufficiency were compared according to their serum TSH, fT3, fT4, total T3, total T4, anti-TPO and anti-Tg levels.Serum TSH, total T3, total T4, fT3, fT4, anti-TPO, anti-Tg and 25(OH)D3 levels were measured in venous blood samples. TSH, total and fT4, total and f T3, anti-TPO and anti-Tg were measured by chemiluminescence method using the autoanalyzer Architect i2000sr .For measuring 25(OH)D3, venous blood samples were collected into plain tubes, serum was separated and stored at -70 ˚C for a week until analysis. 25(OH)D3 vitamin levels were measured by the HPLC method using a Shimadzu analyzer . The intra- and inter-assay coefficients of variation for 25(OH)D3 were 5.6 and 8.0, respectively. The lower detection for 25(OH)D3 was 4 ng/mL (10 mmol/L). No external standards were used.All statistical analysis were performed by using the software SPSS for Windows V16.0. Vitamin D level distribution was compared with chi-square test between patients and the control group. Seasonal homogeneity of the drawn blood samples of the individuals with HT and the controls were confirmed by chi-square test. Odd ratios between HT and serum 25(OH)D3 levels were calculated by using one variable logistic regression. Comparing means between groups of more than 2 were done by using the Kruskal-Wallis method. Comparison between 2 groups was performed by Mann-Whitney U-test. By using Bonferroni correction, a p-value of 0.016 was accepted as significant between 2 groups. Nonparametric correlations were performed by using Spearman’s test. The data are expressed as means ± standard deviation. A p-value below 0.05 was considered as statistically significant.Demographic characteristics of the patients with HT and the control group are presented in Euthyroidism, subclinical hypothyroidism, OHP and subclinical hyperthyroidism were diagnosed in 51 (56.6%), 30 (33.3%), 7 (7.77%) and 2 (2.2%) of the patients. None of the patients had overt hyperthyroidism. Because of the low numbers, patients with OHP and subclinical hyperthyroidism were omitted from statistical analysis. Patients with subclinical hypothyroidism and euthyroidism were similar in terms of age and sex distribution. 25(OH)D3 levels were not different among patients with euthyroidism and subclinical hypothyroidism. 25(OH)D3 levels of the euthyroid patients were significantly lower than those of the control group (p=0.005) .In the total group of patients, serum 25(OH)D3 level was positively correlated with serum total T4  . There wThe results of the present study demonstrated that patients with HT had significantly lower serum 25(OH)D3 levels compared with healthy controls and that the prevalence of 25(OH)D3 deficiency was higher in HT subjects. Recently, the effects of vitamin D on autoimmune diseases have come to attention. While some clinical studies have shown a relationship between autoimmune thyroid diseases and serum vitamin D levels, others have not. In a study on 161 adult patients with HT, 25(OH)D3 levels were reported to be lower and prevalence of 25(OH)D3 insufficiency (<30 ng/mL) to be significantly higher in the patients than the 162 controls . In a stMoreover, in adult patients with HT, newly diagnosed and euthyroid both with no treatment (n=180) and in those previously diagnosed and on LT4 treatment (n=180), serum 25(OH)D3 levels were found to be lower than those in the control group (n=180), while 25(OH) D3 deficiency severity was correlated with duration of HT, thyroid volume and antibody levels . In a stThese conflicting results can be partly related to the variations in study designs and in the definitions of vitamin D deficiency and in a lack of consensus regarding optimal serum 25(OH)D3 levels among healthy subjects. Thus, it is not easy to draw a conclusion about whether vitamin D deficiency is a risk factor for the development of autoimmune thyroiditis.From a different point of view, it has been postulated that the low levels of serum 25(OH)D3 seen in autoimmune disease is a secondary phenomenon which reflects down regulation of vitamin D metabolism rather than being a causal factor leading to illness. Vitamin D receptor (VDR) dysfunction is proposed to be the key factor for the disease process . Once 1.In the current study, although the prevalence rates of 25(OH)D3 insufficiency and sufficiency were not different in patients and controls, the prevalence of 25(OH)D3 deficiency was significantly higher in the HT patients. It was interesting to note that although statistically insignificant, the incidence of 25(OH)D3 insufficiency was higher in the control group. Vitamin D insufficiency was also encountered in our control group, but the patients with HT had vitamin D levels even lower. Based on the results of our study, we suggest that a serum 25(OH)D3 level of 20 ng/mL can may be accepted as a critical cut-off level.Thus in individuals with serum 25(OH)D3 <20 ng/mL, HT was observed 2.28 times more frequently as compared to ones with higher 25(OH)D3 levels. A recent study evaluating the effects of vitamin D in immune modulation showed a direct correlation of vitamin D levels with T regulatory cells and an inverse correlation with T effector cells, which are both related in the pathogenesis of autoimmune diseases. These authors suggested that there is an optimal vitamin D level to control immune regulation and pathogenic resistance . ClinicaBesides affecting the thyroid gland through immune-mediated processes, vitamin D has also been shown to affect thyroid functions. In rat thyroid cells, vitamin D attenuates TSH-stimulated iodide uptake and cell growth . VitaminIn conclusion, this study showed that possibly there is an association between vitamin D and HT, especially in individuals with 25(OH)D3 levels lower than 20 ng/mL. The mechanism for this association and whether it is a cause or effect relationship is not clear.