The aim of this study was to identify patients not requiring ureteric stone surgery based on pre-operative imaging (within 24 hours) prior to embarking on semirigid ureteroscopy (R-URS) for urolithiasis.The imaging of all consecutive patients on whom R-URS for urolithiasis was performed over a 12-month period was reviewed. All patients had undergone a plain x-ray of the kidney, ureters and bladder (KUB), abdominal non-contrast computed tomography (NCCT-KUB) or both on the day of surgery.A total of 96 patients were identified for the study. Stone sizes ranged from 3mm to 20mm. Thirteen patients (14%) were cancelled as no stone(s) were identified on pre-operative imaging. Of the patients cancelled, 8 (62%) required NCCT-KUB to confirm spontaneous stone passage.One in seven patients were stone free on the day of surgery. This negates the need for unnecessary anaesthetic and instrumentation of the urinary tract, with the associated morbidity. Up-to-date imaging prior to embarking on elective ureteric stone surgery is highly recommended. Urolithiasis has a prevalence of 1–2% in the general population,Ureteric stone disease often presents with symptoms of ureteric colic , loin to groin pain, nausea and sometimes vomiting. A history of previous stone disease should also be ascertained. Diagnostic imaging should be obtained to support the clinical diagnosis. Helical non-contrast computed tomography of the kidney, ureters and bladder (NCCT-KUB) has become the gold standard for the radiological diagnosis of urolithiasis.Ureteric calculi can pass spontaneously. The size, site and shape are determinant factors in the rate of spontaneous stone passage. For smaller stones, where the spontaneous passage rate is high , a conservative approach is generally the initial management with intervention for those that fail to pass.The need for pre-operative imaging to confirm the ongoing presence of calculi was the subject of some debate at the British Association of Urological Surgeons Section of Endourology meeting in Norwich in 2009. An informal discussion of the floor during a live surgical procedure confirmed that some units have abandoned pre-operative imaging for ureteric stone cases.Semirigid ureteroscopy (R-URS) is not without a small but significant risk of morbidity such as sepsis (1%) and ureteric stricture (1–2%).The aim of this study was to identify the number of patients not requiring R-URS stone surgery following pre-operative imaging as well as the imaging modalities used to determine when surgery is no longer required.All patients listed for R-URS for ureteric calculi over a 12-month period from June 2008 to June 2009 were identified from the theatre Operating Room Management Information System (ORMIS) register. Diagnostic imaging at presentation was reviewed in all patients to determine the size, site and number of ureteric calculi, as was any imaging in the interim period prior to surgery. All patients admitted for elective R-URS underwent either a plain x-ray of the kidney, ureters and bladder (KUB) or NCCT-KUB or, occasionally, both on the day of proposed surgery. This was established practice in our institution at the time of the study. KUB was performed for radio-opaque calculi and NCCT-KUB for radiolucent calculi. All imaging was reviewed by the operating surgeon prior to theatre. All patients cancelled within 24 hours of surgery as a result of no stones on their imaging were identified from the ORMIS register.In total, 96 patients were listed for R-URS for ureteric calculi. Stone sizes ranged from 3mm to 20mm, with a median size of 7mm. In 28 cases (29%), stones were radiolucent on plain KUB but were seen on NCCT-KUB at the time of diagnosis, thus necessitating NCCT-KUB on the day of admission. Pre-operatively, 57 patients (59%) had plain KUB only, 29 (30%) had NCCT-KUB only and 10 (10%) had both.Overall, 13 patients (14%) were cancelled within 24 hours prior to elective surgery due to no stones being identified on pre-operative imaging. Stone sizes in the spontaneous passage group ranged from 3mm to 10mm , with a A significant proportion of patients were found to have passed their stone(s) spontaneously as discovered on pre-operative imaging prior to elective surgery. This can occur in a wide variety of stone sizes and location.In our institution, plain KUB and NCCT-KUB are performed under standard protocols. The radiation exposure for a single KUB is 0.7mSv and for NCCT-KUB it is 5mSv.The cumulative mean radiation exposure following pre-operative imaging was 9.43mSv (95% CI: 8.69–10.20mSv). An online radiation exposure calculator was used to assess the additional risk of cancer based on age, sex and imaging modalities along with the radiation dose for each modality.A negative R-URS carries with it a small but significant risk of morbidity with complications being reported in up to 6% of cases,In total, 17 patients underwent interim percutaneous nephrostomy (PCN) tube insertion and 4 underwent ureteric stent insertion under fluoroscopy guidance between acute presentation and planned R-URS. Of those 39 patients who underwent pre-operative NCCT-KUB, 10 had PCN tubes in situ and 2 had ureteric stents in situ. Spontaneous stone passage occurred in one patient with a PCN in situ and, consequently, R-URS was not performed. The two patients with ureteric stents underwent R-URS as their stones remained present on pre-operative imaging.Of the 83 patients who proceeded to R-URS, 5 (6%) underwent unplanned NCCT-KUB on the day following KUB to confirm the presence of their calculi. Sensitivity of plain KUB has been reported as low as 54%.Patients were treated on the basis of their pre-operative imaging on the day of surgery. If this did not demonstrate any evidence of calculi, patients did not undergo R-URS, irrespective of symptoms, to avoid the risk associated with urinary tract instrumentation in the absence of identifiable calculi. In cases where no stones were found on R-URS, without adequate pre-operative imaging, some may have proceeded to flexible ureteroscopy (F-URS) for fear of the stone having refluxed up into the kidney, involving increased instrumentation and anaesthetic time.This study was performed in a tertiary referral centre. Some cases were complex due to hostile anatomy such as severe scoliosis and congenital dysplasia of the hip. If any uncertainty arose on pre-operative KUB, further NCCT-KUB was requested if necessary, often after consultation with a uroradiologist. These cases had pre-operative NCCT-KUB to ensure clear delineation of their anatomy as well as confirming the presence of a stone.Operating lists are coordinated accordingly to allow time for patients to undergo planned pre-operative imaging. Plain KUB can be performed promptly before operating lists are due to commence. Those patients requiring NCCT-KUB are placed further down the lists to allow time for imaging to be performed and reviewed. If additional imaging is required, prompt communication and a good working relationship with the radiology department ensures there is no delay in the progression of the operating list. Reordering of the lists can occur without undue disruption.A patient known to have right ureteric and left renal calculi was admitted for right R-URS and stone fragmentation. However, pre-operative imaging did not demonstrate the right ureteric calculus. The patient instead proceeded to have F-URS and stone fragmentation for the known left renal calculus.Pre-operative NCCT-KUB for a radiolucent ureteric calculus had shown this had migrated proximally up to the pelvic ureteric junction. F-URS was therefore required.A patient with two radiolucent ureteric calculi at diagnosis demonstrated a single calculus only on pre-operative NCCT-KUB imaging.A patient with a single ureteric calculus at diagnosis demonstrated three ureteric calculi on pre-operative NCCT-KUB.Other benefits were found on performing thorough pre-operative imaging:In the above cases, the treatment plan was altered and different techniques were used to treat their stones. Prior notice could thus be given to theatre staff in readiness of scopes and equipment as well as informing the anaesthetist. This ensures efficient use of theatre time and running of theatre lists. Pre-operative imaging also confirms stone number. In this study, the stone number for two patients changed between initial presentation and elective surgery. This avoided pursuing calculi that had passed spontaneously prior to surgery as well as the premature completion of surgery due to the presence of calculi unknown to the operating surgeon, which would have remained untreated.Pre-operative imaging is essential in the surgical management of ureteric stone disease. One in seven patients (14%) was found not to have stones on pre-operative imaging. Of those patients cancelled, 8 (62%) required NCCT-KUB to prove stone passage had occurred within 24 hours prior to surgery, confirming the value of NCCT-KUB in the pre-operative assessment. We feel this is safer than relying on negative KUB. This avoids unnecessary anaesthesia and instrumentation of the urinary tract, with the associated potential morbidity. Up-to-date imaging prior to embarking on elective ureteric stone surgery is highly recommended.


Soft tissue necrobiosis and T-lymphocyte infiltration within the periprosthetic soft tissue have been linked to a suggested hypersensitivity reaction of the delayed-type following the metal-on-metal arthroplasty. While we observed both synovial necrobiosis and lymphocyte infiltrates in synovial tissues with failed metal-on-polyethylene prostheses, we hypothesized that both findings are unspecific for metal-on-metal bearing coupes. Thus, we wished to quantify the extent of necrobiosis and the amount of T-lymphocyte infiltration in aseptically loosened metal-on-polyethylene prostheses.We analyzed 28 consecutive synovial biopsy specimens obtained at revision surgery of aseptically loosened metal-on-polyethylene prostheses (19 hips and 9 knees) and quantified both the extent of necrobiosis vertically and the density of CD3+, CD4+, and CD8+ lymphocytes within the joint capsular tissue. We excluded patients with inflammatory skeletal disease or with a history of metal hypersensitivity.We found necrobiosis in 23 of 28 cases and it was most often connected with the superficial portions of the synovium. Necrobiosis of deeper tissues was seen in 8 specimens and it was strongly associated with superficial necrobiosis. While CD3+ lymphocytes were detected in each biopsy, 4 cases with more than 300 CD3+ lymphocytes were identified in the group of 26 cases that presented with more than 100 CD3+ lymphocytes within one high-power field. 16 cases with more than 100 CD3+ lymphocytes also showed concomitant superficial necrobiosis of the synovium. In the inflammatory infiltration of periprosthetic synovium, CD8+ lymphocytes predominated over CD4+ cells.Synovial necrobiosis and infiltration of T-lymphocytes are common findings in tissues around aseptically loosened metal-on-polyethylene arthroplasty in patients without a clinically suspected metal hypersensitivity reaction. Thus, neither necrobiosis nor infiltration of T-lymphocytes should be considered to be specific for failed metal-on-metal bearings or metal hypersensitivity reaction. The re-introduction of metal-on-metal (M-o-M) bearings has been associated with adverse biological reactions that appear to be related to metal hypersensitivity reactions. Solid periprosthetic masses showing necrotic granulomatous pseudotumors , necrobiTo determine how often the histopathological findings suggestive of the delayed-type hypersensitivity reaction to metal in M-o-M arthroplasty can also be observed in the synovium obtained from joints with aseptically loosened metal-on-polyethylene (M-o-PE) prostheses, we asked: (1) what is the frequency and morphological pattern of necrobiosis in these cases, and (2) what is the prevalence and number of periarticular soft tissue T-lymphocytes in these specimens?We retrospectively analyzed 28 consecutive synovial biopsies obtained at revision surgery for aseptic loosening of M-o-PE arthroplasties operated by the senior author (WR) from January to June 2008. All implantations had been performed for primary osteoarthritis. We excluded patients with a history of metal hypersensitivity (contact dermatitis) and/or rheumatoid disease. 11 of the patients were men  and 17 were women . 19 specimens were from the hip , and 9 were from the knee . All procedures were unilateral. Infection was not suspected, either clinically (negative radiographic and laboratory findings) or microscopically (negative finding of neutrophilic polymorphonuclear leukocytes in the biopsy specimen).Immediately after the excision biopsy of synovial tissue, the specimens were fixed in formalin and sent to the laboratory for histopathological analysis. They were analyzed macroscopically, photographed, cut in a plane vertical to the synovial surface, and embedded in paraffin wax. The histopathological slides were stained using the hematoxylin and eosin, elastica van Gieson, and chloracetate esterase staining methods. All specimens were also analyzed under polarized light. Immunohistochemical staining was performed simultaneously with monoclonal mouse anti-human antibody against the T-lymphocyte marker CD3 , CD4  and CD8 . The immunohistochemical analysis was validated using positive controls (tonsil) and negative controls (by omitting the primary antibody).Necrobiosis was defined as regressive soft tissue change showing the ghost outlines of histiocytes in areas with maintained collagen structure. Under polarized light, birefringent wear particles were apparent within the lesions, often within or adjacent to ghost outlines of formerly visible histiocytes, and/or associated with the loss of former cell outlines. Both histiocytes and fibroblasts showed degenerated or absent nuclei . StainedVillous necrobiosis  was diag2)  .The results are expressed as median and interquartile range (IQR). For comparison of the nominal variables, Fisher's exact test was used. The depth of necrobiosis and the number of T-lymphocytes were not normally distributed and were analyzed using the non-parametric Mann-Whitney test. Correlations were considered significant when the p-value was less than 0.05. All data were analyzed using SPSS software version 18.0.Necrobiosis was detected in 23 of 28 synovial biopsies: 13 specimens showed necrobiosis of the villous synovial projections, 17 cases showed superficial necrobiosis, and 8 biopsies had deep synovial tissue necrobiosis . NecrobiCD3+ lymphocytes were detected in each specimen (median number of CD3+ cells per high-power field (HPF): 176; IQR: 129–260). While none of the patients showed less than 50 CD3+ lymphocytes per HPF and only 2 patients had less than 100 CD3+ lymphocytes per HPF, 4 specimens had more than 300 CD3+ lymphocytes per HPF. 3 of the 4 patients who had more than 300 CD3+ lymphocytes per HPF were men. The number of CD3+ lymphocytes was not statistically significantly different between those cases without superficial necrobiosis  and cases with positive findings . In addition, the number in men  was similar to that in women . One of these 4 biopsy specimens with more than 300 CD3+ lymphocytes per HPF did not show necrobiosis. When compared with failed hips , soft tissue specimens from knee joints with aseptically loosened M-o-PE arthroplasty did not have a substantially different number of T-lymphocytes . It is worthy of mention that superficial necrobiosis was detected histopathologically in 16 cases that also presented with more than 100 CD3+ lymphocytes. Finally, we did not find any significant differences in the number of lymphocytes in the cases with metallosis  compared to the cases with negative findings  .The infiltration of T-lymphocytes involved both CD4+ and CD8+ cells, which were detectable immunohistochemically in every specimen. We did not find any statistically significant differences between CD4+ and CD8+ T-lymphocyte counts (separately) according to anatomic location, whether or not there was metallosis, or whether or not there was necrobiosis .Although necrobiosis and T-lymphocyte infiltration have been observed in failed M-o-M cases suggestive of delayed-type hypersensitivity to metal, the specificity of such findings has been considered to be uncertain. In fact, most reports on this issue described single cases or small groups of cases, and seldom considered control tissues from non-M-o-M failures. In our routinely diagnosed cases, we have observed both necrobiosis and lymphocyte infiltration of periprosthetic synovial soft tissues. Thus, we wanted to investigate the morphological patterns of necrobiosis and the amount of T-lymphocyte infiltration in retrieved M-o-PE prostheses revised for aseptic loosening and we conducted the current retrospective observational morphological study.We observed necrobiosis in the majority of study cases. Although the diagnosis of necrobiosis did not appear to be problematic within villous projections or deeply localized lesions, it was more of a problem in a few cases within the areas with fibrinous effusion attached to the synovial surface that were also associated with the loss of synoviocytes. In these cases, staining with elastica van Gieson was helpful for differentiation between fibrinous exudates and necrotic tissue. While all cases with deep soft tissue necrobiosis also showed superficial necrobiosis, we hypothesized that such areas might represent folding of the synovial surface with large or irregularly deep invading superficial necrobiosis promoted by local anatomic variations in the structure and elasticity of the adjacent soft tissue. One can only speculate whether such propagation of superficial necrobiosis into deeper soft tissue also plays a substantial role in the pathogenesis of the anterior localized soft tissue granulomatous pseudotumors , b folloAlthough lymphocyte infiltration of periprosthetic tissues was related to adverse biological reactions following M-o-M arthroplasty, there is an increasing amount of data on the prevalence of lymphocyte infiltration in non-M-o-M failures. For the purposes of the current study, we did not differentiate between the diffuse and perivascularly located lymphocytes but counted the number of CD3+ lymphocytes per HPF in areas with maximum infiltration in order to overcome the limitations of descriptive grading systems. Based on our results, it seems probable that a cut-off at 100 lymphocytes per HPF is a relatively low value for consideration of the lymphocyte infiltration to be excessive. Indeed, in our earlier study on morphological findings in M-o-M hip resurfacing revised for groin pain , we founEven though we specifically focused on the number of T-lymphocytes, this particular inflammatory cell population was present in all cases. However, even if we speculate that some of the cases were patients with asymptomatic metal hypersensitivity, this result was rather unexpected and we did not find any explanation other than the fact that T-lymphocytes are a well-known to be a player in the periprosthetic cellular immune response in aseptic loosening of M-o-PE arthroplasties in patients with no history of rheumatoid disease or metal hypersensitivity.We recognize that our study had several limitations. First, because the exact anatomic location of the biopsy could not be assessed retrospectively, we ignored the possible anatomic variability in the layering of the synovial membrane and capsular soft tissue both within each joint and between knee and hip joints. However, it is possible that the extent of necrobiosis and the soft tissue invasion of wear particles are substantially affected by the variable thickness of loose soft tissues between the less elastic fibrous joint capsule and synovial surface. For the same reason, we could not exclude potential entrapment or impingement of the periarticular soft tissue that might also contribute to ischemic changes in the villous synovial projections. Secondly, because it is generally accepted that T-lymphocytes are pathogenetically associated with delayed-type hypersensitivity , we did Based on our results, we suggest that both synovial necrobiosis and T-lymphocyte infiltration are common findings in tissues around aseptically loosened metal-on-polyethylene arthroplasty in patients without any clinically suspected metal hypersensitivity reaction. Thus, neither necrobiosis nor T-lymphocyte infiltration should be considered specific for failed metal-on-metal bearings or metal hypersensitivity reaction.


Dear Sir,The risk of postoperative complications such as fistula recurrence or incontinence increases in patients with recurrent fistulas and high transsphincteric and suprasphincteric fistulas , 2. Due ® III, Biomet Merck, UK) constitutes 10 % of the original volume of the collected blood and more than 90 % of its mass is made of platelets. Platelet-rich plasma preparation is administered to the fistula tract following curettage  is platelet concentration derived from centrifuged full blood collected from a patient right before surgery. The platelet-rich plasma (PRP) obtained using the Gravitational Platelet Separation III System  was administered to the narrow lateral tract following curettage. The patient was discharged home on postoperative day 3. No recurrence of the fistula was reported within the 6-month follow-up period.A 37-year-old male with recurrent intersphincteric anterior fistula-in-ano, and with a narrow, transsphincteric branch running towards the puborectalis loop also underwent surgical treatment at our unit. At a time of surgery, the main tract of the fistula was removed and APRP (GPSThe first experiences with using growth factors were related to the treatment of patients with chronic skin ulcers . At presIn the healing process, thrombin stimulates platelet alpha-granules to release growth factors which, through appropriate stem cell receptors, stimulate stem cell differentiation.Reports of using autologous growth factors for the treatment of fistula-in-ano date back to the late 1990s. In 1999, a paper on the treatment of patients with recurrent fistula-in-ano was published . It descIn the literature, there are reports of using APRP as an element of multi-stage treatment of fistula-in-ano. The first stage involves inserting a loose seton through the fistula tract in order to drain and heal any local inflammatory lesions. The second stage involves APRP administration, which resulted in fistula healing in as many as 75 % of patients . Hagen eAvailable publications include papers that present small groups of patients, different types of fistulas qualified for APRP treatment and different methods of surgery. However, the results are encouraging since this method has no negative effect on anal sphincter function.


Drosophila species, the correlation between their phylogenetic classification and the classification on the BLSOMs was observed to visualize oligonucleotides diagnostic for species-specific clustering.A strategy of evolutionary studies that can compare vast numbers of genome sequences is becoming increasingly important with the remarkable progress of high-throughput DNA sequencing methods. We previously established a sequence alignment-free clustering method “BLSOM” for di-, tri-, and tetranucleotide compositions in genome sequences, which can characterize sequence characteristics (genome signatures) of a wide range of species. In the present study, we generated BLSOMs for tetra- and pentanucleotide compositions in approximately one million sequence fragments derived from 101 eukaryotes, for which almost complete genome sequences were available. BLSOM recognized phylotype-specific characteristics  in the genome sequences, permitting phylotype-specific clustering of the sequences without any information regarding the species. In our detailed examination of 12  Genome sequences, even protein-noncoding sequences, contain a wealth of information. The G + C content (%GC) is a fundamental characteristic of individual genomes and is used for a long period as a basic phylogenetic parameter to characterize individual genomes and genomic portions. The %GC, however, is too simple a parameter to differentiate wide varieties of genomes. Many groups have reported that oligonucleotide composition varies significantly among genomes and can be used to study genome diversity –8. BecauThe unsupervised neural network algorithm known as Kohonen's Self-Organizing Map (SOM) is a powerful tool for clustering and visualizing high-dimensional vectorial data on a two-dimensional map –11; olig Drosophila in detail, for which the genomes of many closely related species have been sequenced. We constructed BLSOM with tetra- and pentanucleotide compositions in most  of the Drosophila genomes available and focused on species-specific characteristics of oligonucleotide frequencies in each Drosophila genome (genome signature), in connection with their phylogenetic classification.To test the power of BLSOM to detect differences among eukaryote genomes and particularly among closely related species, the present study examinedtakaabe@ie.niigata-u.ac.jp or y_wada@nagahama-i-bio.ac.jp).SOM is an unsupervised neural network algorithm that implements a characteristic nonlinear projection from the high-dimensional space of input data onto a two-dimensional array of weight vectors –11. We mhttp://hgdownload.cse.ucsc.edu/downloads.html. When the number of undetermined nucleotides (Ns) in a sequence exceeded 10% of the window size, the sequence was omitted from the analysis. When the number of Ns was less than 10%, the oligonucleotide frequencies were normalized to the length without Ns and included in the analysis.Genome DNA sequences were obtained from A large number of genomes, including a wide variety of eukaryotes, have been sequenced with the remarkable progress of currently available DNA sequencing technologies. To investigate the clustering capacity of BLSOM for sequences derived from a wide range of eukaryotes, we first analyzed tetra- and pentanucleotide frequencies in ca. 1,800,000 nonoverlapping 5 kb sequences as well as ca. 900,000 nonoverlapping 10 kb sequences and overlapping 100 kb sequences with a 10 kb sliding step from 101 eukaryotic genomes, most of which were completely sequenced. To analyze sequences derived even from lower eukaryotes with small genome sizes in accurate detail, excess representation of higher eukaryotes' sequences derived from their large genomes had to be avoided. Therefore, for higher eukaryotes, 100 kb sequences were selected randomly from each large genome up to 200 Mb and used for the BLSOM analyses. In DNA databases, only one strand of a pair of complementary sequences is registered, and the choice between the two complementary sequences is often arbitrary in the registration. When global characteristics of oligonucleotide composition in the genome are considered, the distinction of frequencies between the complementary oligonucleotides  is not important in most cases. In the present study, BLSOM was constructed with frequencies for degenerate sets in which the frequencies of a pair of complementary tetra- or pentanucleotides were added . It should be stressed that complex combinations of many oligonucleotides contributed to the self-organization of sequence fragments according to phylotype and that BLSOM could visualize the diagnostic oligonucleotides in an easy-to-understand manner.BLSOM provides a powerful ability for visualizing diagnostic oligonucleotides that contribute to the self-organization of sequences according to phylotype , 15. The Drosophila genomes were specifically marked in pink on the BLSOMs . This showed that BLSOM could properly extract sequence characteristics of Drosophila genomes with phylogenetic clustering through sequence homology searching. The diagnostic oligonucleotides contributing to the clustering according to the Drosophila group on BLSOMs could be assigned as shown in In e BLSOMs . In Figu Flybase . Distrib Drosophila genomes. Nodes containing sequences only from one Drosophila group listed in The results presented in Figures In When characteristic oligonucleotides, both underrepresented and overrepresented in each genome, are considered Figures  and 4, vFor almost half of genes from the novel genomes sequenced, it has become clear that protein functions cannot always be estimated through sequence homology searching. We have applied BLSOM to protein sequence studies to analyze the frequency of oligopeptides and found the separation (self-organization) of proteins according to their functions . This fiSupplemental Figures list the distribution patterns of each pair of complimentary tetra- and pentanucleotide in 100-kb DegeTetra- and DegePenta-BLSOM for the overlapping 100-kb sequences of 101 eukaryotic and 12 Drosophila genomes.Supplementary Figure S1. Level of each pair of complimentary tetranucleotide in 100-kb DegeTetra-BLSOM for the overlapping 100-kb sequences of 101 eukaryotic genomes.Supplementary Figure S2. Level of each pair of complimentary pentanucleotide in 100-kb DegePenta-BLSOM for the overlapping 100-kb sequences of 101 eukaryotic genomes.Supplementary Figure S3. Level of each pair of complimentary pentanucleotide in 100-kb DegePenta-BLSOM for the overlapping 100-kb sequences of 12 Drosophila genomes.Click here for additional data file.


To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells.We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation. The material composition and structure of bone determine the strength and elasticity of bone to tolerate a load, and the modulate loads on bone strengthen its structure. This adaptation is carried out by the cellular machinery of bone remodeling. Bone remodeling functions in damage repair; and therefore, the adaptation of structure to loading is anticipatory damage prevention et al. developed a rat tibia model under 4-point bending et al., a rat ulna model under axial compression et al.et al.et al. developed a noninvasive 3-point bending loading method with programmed wave forms for use on rat tibiae, and observed a relationship between bone formation and mechanical loading For investigation of the function of this adaptive nature, various apparatuses of mechanical loading have been developed. For example, Turner in vitro, the mechanical stress causes remodeling of cell-matrix adhesions, in which the cytoskeleton rapidly responds to external force by actin assembly in vivo, although woven bone and lamellar bone formation is enhanced by mechanical loading on bone surface These methods mainly caused woven bone formation on bone surface as a primary response to the applied loads. The mechanical strain must be signaled to the bone surface, i.e., periosteum which is assumed to be responsible for sensing the mechanical strain. The periosteum is a membrane that lines the outer surface of all bones, except at the joints of long bones. This membrane, which consists of dense irregular connective tissue, is divided into an outer fibrous layer and an inner osteogenic layer. The fibrous layer contains fibroblasts, whereas the osteogenic layer contains the progenitor cells that develop into osteoblasts. In the observation of molecular and cellular phenomena acted by mechanical stress in-situ fluorescence imaging. This imaging method enabled us to observe the quick changes in periosteal cell shapes by mechanical loading. Upon detailed analysis, we observed that the mechanical loading rapidly decreased the quantity of stress fibers of the actin cytoskeleton and changed the nuclear shapes in the periosteal cells, and then disorganized actin cytoskeleton was remodeled in a time-dependent manner. Moreover, by treatment with cytochalasin-D, which disrupts the actin cytoskeleton, periosteal hypertrophy was induced. Furthermore, by intravenously injecting with a ROCK inhibitor, which inhibits polymerization of the actin cytoskeleton, the mechanical loading-mediated periosteal hypertrophy was reduced. Taken together, we demonstrated that actin disorganization and subsequent remodeling were linked to the mechanical stress-mediated hypertrophy of the periosteum, which promoted bone formation.In the present study, in order to investigate the cellular and molecular mechanism of mechanotransduction in the mechanically loaded periosteum, developed noninvasive 3-point bending loads with programmed waveforms were applied to mouse tibiae. Since programmed waveforms consist of 2 types, i.e., short-pulse and long-pulse, we selected the latter as giving the less inflammatory signals. After producing a load on the mouse tibial periosteum, we observed hypertrophy of the periosteum. In the hypertrophic periosteum, cell proliferation and osteoblast activation were detected. To investigate the relationship between programmed loading and periosteal hypertrophy, we further devised a new observation method named A 3-point bending bone formation experimental system was established earlier for the rat tibia As shown in in-vivo experiments for the short-pulse and long-pulse mode. The strain induced in the mouse tibia by the dynamic loading was measured with strain gauges. The peak strain on the surface around the loading point was approximately 2100 µε. This strain was equivalent to that observed in the previous investigations on mechanically induced bone formation using mouse model The C57BL/6 mice were divided into 2 groups. The first group received a loading, with the waveform designed as illustrated in We show the time schedule for loading (red arrow) and calcein labeling (green triangle) in We also assessed the level of inflammation caused by the loading, by examining the expression of Mac-3, which is a marker of the macrophages that penetrate into inflamed tissue . In the To investigate the effects of the stress change toward compression on the periosteum, we histologically analyzed this outer layer of bone. After loading had been applied to the left tibia in the long-pulse condition, we performed hematoxylin-eosin (H&E) staining of the left tibia at days 1,3,5, and 7 , and of To confirm the cell proliferation in the long-pulse mode, we performed immunostaining using an anti-Ki-67 antibody, since Ki-67 is a marker of proliferating cells. In the loaded left tibia, Ki-67-positive cells were detected at the side opposite to the loading point treated with the long-pulse at day 3 , and becNext, to identify osteoblasts in the hypertrophic periosteum, we performed staining for alkaline phosphatase (ALP), a marker of osteoblasts. Although ALP is a ubiquitous plasma membrane-bound enzyme synthesized at various levels by many cell types, high levels of ALP are found in osteoblasts and preosteoblasts in bone Since we observed woven bone formation in response to the stress change toward compression, we investigated the induction of osteoblast differentiation in periosteal cells by analyzing the expression of BMP-2, which is a typical differentiation factor involved in the differentiation of mesenchymal cells into osteoblasts. We performed Western blot analysis on lysates of periosteal cells taken from the side opposite to the loading point from the long-pulse treated left tibia and the control right tibia at day 3. Anti-BMP-2 antibodies detected bands of a precursor of BMP-2 , arrow ain situ immunofluorescence imaging, to visualize the rapid appearance of molecules and quick cytoskeletal alterations in the periosteal cells. The schematic of this method is shown in in vivo. We excised tibiae of mice injected intraperitoneally with calcein 2 days before sacrifice, fixed the whole tibiae, and then immunofluorescently stained them. The calcein labeling on the bone surface enabled us to distinguish the periosteum from the other connective tissues. Firstly, we observed the cytoskeletal actin network in periosteal cells on the bone surface ; and the ROCK inhibitor or the isotonic sodium chloride solution as a vehicle-injected control was injected 4 times (at days 1–4) via a tail vein. The mice were then sacrificed at day 5, and observed for periosteal thickness in the same manner described above. In the vehicle-injected mice, the periosteal thickness was about 120 µm. The results showed that the thickness of the periosteum was slightly decreased in mice injected with 25 mg/kg of the ROCK inhibitor . Howeverin vivo models of mechanotransduction. In this regard, the non-invasive loading on bones of experimental animals, axial compression in-situ immunofluorescence imaging.In development of researches for mechanical stress, there was a great need for noninvasive in-situ immunofluorescence imaging, we have succeeded in noninvasive loading and quick observation of the periosteum. The results showed that the actin fibers were disorganized soon after loading, and the disorganized actin fibers were remodeled 2 days after loading to induce periosteal cell proliferation. This periosteal hypertrophy induced by cell proliferation led to the formation of woven bone. Since the tissue change in the periosteum after loading was very quick, our 2 newly developed methods were very useful to detect molecular changes without the interference of inflammation. Nevertheless, it was very difficult to catch the first actin disorganization inducing event triggered by the loading. The organization of the actin cytoskeleton may be part of the sensing apparatus used by cells to detect and respond to mechanical signals. In vitro, reorganization of the actin cytoskeleton in osteoblasts into stress fiber bundles is required for fluid shear-induced expression of genes, cyclooxygenase-2 and c-fos, linked to mechanotransduction in bone formation With coupling the new bending method, the long-pulse mode, with A specific inhibitor of Rho-dependent kinase (ROCK) suppressed periosteal hypertrophy by inhibiting action reorganization for remodeling. On the other hand, while active RhoA is required for stress fiber formation, its principal downstream effectors are the diaphanous-related formin protein mDia 1 that promotes assembly of actin stress fibers, and ROCK that induces non-muscle myosin II-driven actin contraction Interestingly, we observed alteration of the nuclear shape in the early time of loading (from day 0 to 2). Such abnormal shapes of nuclei were reported to be seen in the Hutchinson-Gilford progeria syndrome (HGPS), which is caused by mutations of the lamin A gene resulting in nuclear stiffness in-vitro experiments. In our stress change toward compression model, we observed mature BMP-2 expression at day 3 after loading. BMP-2 expression is known to be involved in cortical bone healing Actin reorganization into stress fibers is required for the induction of osteogenic cells in Finally, in the aspect of clinical application, decreased fracture toughness in bones from older people is indicative of a change in the quality of bone tissue with age. This reduced bone toughness may partly explain the observation that fracture risk in older people is greater than predicted by the loss of bone mass alone In this study, 10-week-old male C57BL/6 mice were used. All animal experiments were performed following The Regulation on animal experimentation of Tokyo Institute of Technology and Tokyo Medical and Dental University. This study was approved by the Animal Care and Use Committee of Tokyo Institute of Technology (permit number: 2009047) and by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (permit number: 0070175).Rabbit anti-mouse periostin antibodies were described previously S)-4-bromo-5-(2-methylhomopiperazin-1-ylsulfonyl) isoquinoline dihydrochloride, a ROCK inhibitor Inhibition of actin remodeling was performed by tail vein injection of mice with , their tibiae were dissected, fixed in 70% ethanol for 48 hr, and embedded in resin. Serial non-decalcified transverse sections were prepared with a diamond disc saw , and digital fluorescent images of calcein, 1 mm apart, were obtained by using a confocal laser scanning microscope . The images were exported into Image J (NIH) for cropping and linear contrast adjustment.For tissue preparation, mice were sacrificed under anesthesia, fixed with 4% paraformaldehyde (PFA) in PBS by transcardial perfusion. And immediately, the excised their tibiae were immersed in 4% PFA in PBS at 4°C for 20 hr. After a wash with PBS, the samples were decalcified in 500 mM EDTA solution at 4°C for 2 weeks, and then were dehydrated through a graded series of ethanol prior to being embedded in paraffin. Series of 5-µm-thick paraffin sections were prepared for histological analysis or for immunostaining. For immunostaining of Ki-67, the antigen unmasking technique with citrate buffer was performed. For demonstrating the alkaline phosphatase activity, 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazoliumchloride (BCIP/NBT) was used as the chromogen.Mice were fixed with fresh 4% PFA in PBS as described above. After a wash with PBS, soft tissues were dissected, and the excised whole tibia was incubated with 10 µg/ml glycine in PBS to block free aldehyde groups for 10 min, and then with 0.1% Triton X-100 for 5 min at room temperature. After having been blocked with 1% bovine serum albumin (BSA) in TBS (preheated at 56°C for 30 min) for 30 min at room temperature, the whole tibia was incubated with the desired primary antibodies in 1% BSA in TBS for 2 hr at room temperature. Further incubation with an optimal dilution of fluorescence-tagged secondary antibodies was performed for 1 hr, followed by washing and post-fixation with 4% PFA. Cytoskeletal actin was stained with Alexa Fluor 568-labeled phalloidin. Nuclei were stained with TOPRO3 . Immunostained whole tibiae were immersed into dish. Fluorescent images were collected by a laser-scanning confocal microscope equipped with a water-immersion objective lens . The images were imported into Photoshop  for cropping and linear contrast adjustment.For Western blotting of BMP2, periosteal cells were collected from tibia subjected to mechanical loading or drug injection at day 3. These cells were lysed with SDS sample buffer containing 50 mM dithiothreitol, and the lysates were then boiled for 10 min. Equal amounts of samples were separated on 13% SDS-PAGE gels and then transferred to nitrocellulose transfer membranes. Non-specific binding was blocked by immersion of the membranes for 30 min in 5% skim milk in TBS containing 0.1% Tween 20 (TBST) at room temperature. The membranes were washed with TBST, incubated overnight at 4°C with primary antibodies diluted in 1% skim milk in TBST. After having been washed 3 times with TBST, the membranes were incubated with secondary antibodies for 1 hr at room temperature; thereafter, they were washed 3 times with TBST, incubated with Immobilon Western Chemiluminescent horseradish peroxidase substrate (Millipore), and exposed to Fuji medical x-ray film .http://www.r-project.org/), in which statistical significance was assumed for p<0.05.Welch's t tests in combination with Bonferroni-Holm correction for multiple comparisons were performed with the R software  In the right tibia, used as a control, Mac-3-positive cells were rarely detected in either short-pulse or long-pulse group. (B) In the left tibia in the short-pulse group, Mac-3-positive cells were detected around the loading point (arrow).  In the left tibia in the long-pulse group, Mac-3-positive cells were also detected around the loading point , but not at the side opposite to the loading point (D). Non-specific signals were detected in osteogenic layer. Scale bar; 50 µm (A–D), 200 µm (E). Red arrowhead indicates loading direction.(TIF)Click here for additional data file.


Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes  and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas.A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44.In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion. Fibroadenoma represents the most frequent breast lesion in adolescents and young women with the giant fibroadenoma (GF) being an uncommon variant. GFs, which occur mostly in adolescent girls, are characterized by their large size (more than 5 cm). They are encapsulated masses and generally asymptomatic. Their rapid growth (between two and five months) is associated with skin congestion and ocasionally ulceration. It is thought that increased estrogen receptor sensitivity is responsible for the etiology of GF .Aberrant methylation of CpG islands (CpGIs) in promoter regions is known to be involved in the silencing of tumor-suppressor genes, steroid receptors, cell adhesion molecules and cell cycle regulator genes and appears to be an early event in the etiology of breast carcinogenesis . The abein situ , and lobular neoplasia. The malignant transformation of a fibroadenoma is a rare event, with about 100 cases reported in the world literature. Despite this fact we decided to analyze the methylation status of a GF which is a rapidly growing benign breast lesion , O6-methylguanine-DNA methyltransferase [MGMT (-463bp)], Wilms' Tumor-1 [WT-1 (-146bp)], Breast Cancer 2 [BRCA2 (+138bp)] and Hermen Antigen [CD44 (+28bp)] . The surprising novelty, however, is that this finding occurs in a benign lesion.These five aberrant methylated genes play diverse functions in the cell: DNA reparation (MGMT and BRCA2), cell cycle control , proliferation  and cell adhesion CD44). The methylation of three of them  has been widely reported in breast tumors 4. The me. MethylaOur data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case there were no previously published data regarding the role of methylation in GFs, considered by definition to be a benign breast lesion. These findings should be taken into account to evaluate whether it is associated with the different etiology of non-GFs and GFs. Further studies will be necessary to draw more definitive conclusions about the meaning of the methylation de-regulation in this type of disease.BRCA2: Breast Cancer 2; CD44: Hermen Antigen; CpGIs: CpG islands; ERα: estrogen receptor α protein; ESR1: estrogen receptor-α; GF: giant fibroadenoma; MGMT: O6-methylguanine-DNA methyltransferase; MS-MLPA: Methylation-specific multiplex ligation-dependent probe amplification; p16INK4a: Cyclin-dependent kinase inhibitor 2A; WT-1: Wilms' Tumor-1Written informed consent was obtained from the patient's next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The study was approved by the Bioethics Committee of the School of Medical Sciences, National University of Cuyo, Mendoza, Argentina.The authors declare that they have no competing interests.DMM performed the methylation study and revised the manuscript critically. FEG participated in the study design with JO. OT carried out the pathological studies. MR participated in interpretation of data and revised the manuscript critically. LMV-R designed the study and wrote the manuscript. All the authors discussed the results and read and approved the final manuscript.


Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations.We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD , is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering.We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases. Many important cellular processes, such as gene expression regulation and transport, are carried out by protein complexes -3. The iMany protein-protein docking methods have been developed in the past employing various ideas and techniques -20. TypiThe accuracy of docking prediction could improve when a part, even if not all, of protein-protein interface (PPI) residues are known. PPI residues for a pair of interacting proteins can be identified by experiments including point mutation such as the alanine scanning -38, chemPPI residues can be also predicted by computational methods, which capture sequence and structural features of PPI regions . There aHere, we present a novel protein docking algorithm, PI-LZerD , which utilizes imperfect PPI residue prediction for guiding protein-protein docking. PI-LZerD performs iterative improvement of docking results starting from an initial run of docking that uses potentially inaccurate PPI prediction as restraints. The base of the docking algorithm used is the LZerD , which we have developed previously . The ideWe start with brief explanation of the original LZerD pairwise protein-protein docking algorithm . As willGiven a protein tertiary structure, protein surface is constructed and then points are distributed evenly on the surface. Typically, about 1500~2000 points are distributed for a 200-250 amino acid long protein Figure . The geoThe scoring function is a weighted sum of the following terms: van der Waals, where, repulsive and attractive parts of the term are considered separately ; an elecWe modified the LZerD algorithm so that additional information of a PPI region can be used to restrict the docking search space. Figure The PI-LZerD algorithm performs pairwise protein-protein docking prediction using additional information of PPI residues as constraints. The algorithm is illustrated in Figure The 1000 decoys are subject to clustering by considering the similarity of docking interface regions. For a given pair of docking decoys, common atoms between the two PPI regions from the two decoys are selected. Then, the RMSD is computed for the common atoms only when the common atoms share more than 60% of all interface atoms of both PPI sites (if the common atoms do not exceed 60% then the two proteins are not clustered together). We call it the common interface RMSD (ciRMSD) of two docking decoys. The ciRMSD is more suitable for the PI-LZerD algorithm as compared to the conventional coordinate RMSD  or the lOnce the ciRMSD is computed for all the pairs of decoys, 60 decoys are selected by considering the physics-based score and the cluster size of the decoys. First, the decoy with the lowest score  is selected and close decoys with a ciRMSD ≤ 4.0Å are discarded from the pool. This process is repeated until 30 decoys are identified. Next, additional 30 decoys are selected based on the cluster size. For each of the decoys, the number of the other decoys within 4.0Å ciRMSD is computed. Then, the largest cluster (i.e. the center decoy with the largest number of close decoys) is selected. If several clusters with the same size are found, the one which has the center decoy with the lowest physics-based score is selected. All the decoys in the cluster are removed, and the process is repeated until 30 representative decoys are selected. Consequently, 30 decoys are selected based on the lowest energy and 30 more decoys are selected based on the cluster size. It was shown that combining the energy value and the cluster size can find more hits than using a single metric alone . From the resulting docking decoys, the top 1000 lowest energy docking decoys are clustered based on ciRMSD, whose cluster centers are sorted by the physics-based score. Since the modified LZerD is run for each of the 60 decoys, in total of 60 LZerD runs are performed.In addition to the 60 runs of the modified LZerD, we run the original LZerD without using predicted PPI information followed by post-filtering by using the predicted PPI residues (naive-filtering method)  at one end of the PPI site along the axis are removed and the same number of residues are added on the opposite side of the PPI site. Thus, the shifting of PPI regions are done geometrically rather than along the protein sequence  and it may take longer for larger proteins. This timing is reported on a computer with a dual-core 2.1 GHz processor with 8 GB RAM. In addition, the pairwise docking program, LZerD, which is the base of PI-LZerD, is also made available at http://kiharalab.org/proteindocking.The executable program of PI-LZerD for Linux is freely available to academic institutions at our website, An obvious approach to use predicted PPI information for protein docking prediction is to select docking decoys with a PPI site that agrees well to the provided PPI information. This approach, termed as the naive post-filtering method, was tested on datasets with the five different accuracy levels of PPI prediction. In addition to the set of accurate PPI information, we used PPI sites shifted by 5, 10, 12, and 15 residues. For each protein complex with PPI information, we run original LZerD to produce top 1000 scoring docking decoys. Then, for each docking decoy, the fraction of the overlap of residues in the provided PPI information the PPI region of the docking decoy is computed for both ligand and for the receptor proteins, and the average of the two are used for sorting decoys.In Figure Next we examine performance of PI-LZerD on the dataset of simulated PPI predictions. This experiment is for understanding the effect of various levels of inaccuracy in PPI predictions to the docking results. In the later sections we discuss the results using actual PPI predictions on bound and unbound docking cases. The full implementation of PI-LZerD  with both 2.5 Å and 4.0 Å Figures  &6D iRMSWe have further benchmarked PI-LZerD on unbound docking cases. Out of 128 unbound docking cases in the protein-protein docking benchmark dataset 3.0 , 118 casWe observe again the same trend as we observed in the previous experiments: PI-LZerD-2 showed consistently better success rate than the base LZerD at each rank cutoff Figures  &7D. At The same prediction results are categorized according to the three difficulty levels for protein docking assigned by the docking benchmark dataset. The 118 unbound cases contain 87 rigid-body docking cases, 16 medium cases, and 15 difficult cases. In Figure Using this test set, we have also examined the effect of using a different number of decoys in the second round of LZerD run in PI-LZerD. As shown in the illustration of the PI-LZerD algorithm Figure , we use Here we show examples of docking predictions that illustrate difference of PI-LZerD as compared to the base LZerD and the naive post-filtering. The first two examples Figures  &9B are 1BUH). The best predictions within top 50 using PI-LZerD/naive post-filtering/LzerD were 1.03 Å (8)/9.09 Å (24)/9.91 Å (17) iRMSD, respectively. In the parentheses the rank of the decoys are shown. The second example . The best iRMSD predictions by PI-LZerD, the post-filtering, and base LZerD were 3.76 Å, 5.71 Å, and 10.28 Å, respectively. The last one, the complex of human factor VIII and human monoclonal BO2C11 Fab (1IQD), again PI-LZerD-2 identified a near-native pose (an iRMSD of 2.91 Å) Figure . The bashttp://haddock.chem.uu.nl/services/CPORT. The distribution of the accuracy of the PPI site predictions is provided in Figures Finally, we compare PI-LZerD with a recently published related method, CPORT . CPORT tThe performance of docking prediction with CPORT and PI-LZerD are compared in Figures We have developed PI-LZerD, a pairwise docking algorithm that uses imperfect PPI prediction to improve docking accuracy. In the series of experiments, we showed that PI-LZerD successfully improved docking results even when accuracy of PPI information is significantly low. Unlike the post-filtering whose success largely depends on the accuracy of provided PPI information, PI-LZerD can use imperfect PPI prediction to improve prediction by exploring docking poses in the neighborhood of provided PPI prediction. PI-LZerD identifies matches of two proteins at local surface regions that only partially overlap with the provided PPI prediction. In addition, employing two iterations of docking searches (PI-LZerD-2) is shown to be more effective than one round of docking (PI-LZerD-1) because the two iterations enable exploring further from the provided PPI site prediction. Improvement of the average docking accuracy by PI-LZerD over LZerD was observed consistently in the series of benchmark experiments including docking using actual PPI site predictions as well as unbound docking cases.While this work focused on pairwise docking, the same procedure can be applied for multiple protein-protein docking algorithms -100. As BL participated in design, implemented the algorithms, and drafted the paper. DK conceived of the study, participated in its design, and finalized the manuscript. All authors read and approved the final manuscript.Supplemental material for "Protein Docking Prediction Using Predicted Protein-Protein Interface". The file contains following five figures. Figure S1. Selecting decoys by the scoring function and/or by the cluster size. Figure S2. The procedure to compute "simulated" incorrect PPI site predictions. Figure S3. Docking prediction results using shifted PPI regions classified by the sensitivity of the PPI predictions. Figure S4. Docking prediction results using shifted PPI regions classified by the fnat of the PPI predictions. Figure S5. Comparison of prediction results using different numbers of decoys for running the second iteration of LZerD.Click here for file


NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene.In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette , and were clearly distinguishable from clones with two or more NEO copies.We compared the results from Southern blot, routinely used to quantify This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method. Two methods are available for the introduction and modification of mouse genomic DNA sequences: (i) microinjection of one or more transgenes into the pronucleus of a fertilized mouse oocyte, which usually leads to random incorporation into the genome as head-to-tail concatamers of 1-1000 units, or (ii) the use of constructs that undergo a site-specific recombination in embryonic stem cells (ES) in order to disrupt the function of a target gene (knockout) or to mutate a gene (knockin). Modified ES cells are then injected into the blastocyst ,3. HomolNEO cassette in ES clones in which homologous recombination was successfully obtained. Here we describe a screening method using a rapid semi-quantitative real-time PCR, which was validated on ES clones with different NEO copies , previously assessed by Southern blot.One member of our group is responsible for a facility within the Molecular Biotechnology Center in Torino, aimed at the preparation of transgenic mice using recombinant ES cells. In routine work, it became necessary to have a rapid test to exclude the presence of additional copies of the NEO probe of 773 bp was used to evaluate the number of transgenic plasmid insertions.From one of the projects involving the preparation of recombinant mice, we selected 45 genomic DNA extracted from ES clones that then underwent Southern blot screening. DNA extraction was performed using standard phenol-chloroform method . SoutherNEO cassette copy number, we set up an assay based on quantitative real-time PCR (qPCR). The assay was performed with two protocols on an ABI 7500 Fast instrument ; data were analyzed using the 7500 software. The two approaches were: (i) an MGB-based assay (MGB-assay), and (ii) a SYBR Green-based assay (SYBR-assay) (Applied Biosystems). Using the Primer Express software (Applied Biosystems) we designed specific PCR primers to amplify 62 bp of highly conserved sequences between two different NEO cassette-containing plasmids PL451 and PL452 (http://web.ncifcrf.gov/research/brb/recombineeringInformation.aspx). The mouse Rpp30 gene  was used as a reference gene. This gene is orthologous to the human RNaseP, widely used as a copy number reference in qPCR assay. Real-time PCR conditions and primer sequences are reported in Figure As an alternative or complementary method to assess the NEO and Rpp30 assays in a duplex PCR including two internal Taqman-MGB probes (5'-FAM labelled for NEO and 5'-VIC labelled for Rpp30)  Figure . Each saIn the SYBR-assay the two reactions were run in separate wells, using a Fast SYBR green mix (Applied Biosystems). The efficiency of each assay was verified with a standard curve starting from 100 ng of mouse DNA, using four serial dilutions from 1:1 to 1:8. The MGB-assay had 80% efficiency, whereas SYBR-assay gave 93% efficiency.NEO copy verified using Southern blot; these values (named nNEO) were expected to be ~ "1" in the case of a single copy of NEO, ~ "2" in the case of two copies of NEO, and "n" for "n" copies of NEO.Gene copy-number was calculated using the comparative delta-delta Ct method . In eachNEO, and 23 clones had multiple NEO copies. We analyzed all samples using both of the two real-time PCR assays  (range 0.81-1.20) for the MGB-assay and 1.01 ± 0.24 (range 0.80-1.24) for the SYBR-assay  showed a variable number of nNEO from ~2 to > 60 . This method appears to be sensitive enough even for identification of single NEO insertions. Of the two assays tested, the MGB-assay is the least convenient, as it requires two specific and expensive fluorescently-labelled probes, and does not show any practical advantage over the SYBR-assay.Southern blot analysis has long been the reference method for the detection of Approaches based on real-time PCR to validate ES positive clones have been previously described, using absolute quantification of NEO, or a relative quantification to detect deletion of the target gene ,10. Our One possible drawback may be the genomic DNA quality/degradation that needs to be checked in case of non-reproducible data.NEO copy number is a valid alternative tool to Southern blot for the rapid screening of large numbers of ES cell clones during the production of knockout or knockin mouse models.In conclusion, our real-time PCR assay to quantify The authors declare that they have no competing interests.CM and EM carried out laboratory experiments, analyzed data and drafted the manuscript; ET analyzed data and revised the manuscript; AB and AB designed the experiments, analyzed data, drafted and revised the manuscript.All authors read and approved the final manuscript.


Objectives and Methods. The aim of the present study was to evaluate, by means of receiver operating characteristic (ROC) curves, whether cognitive vulnerabilities (CV), as measured by three well-known instruments , independently discriminate between subjects with different severities of depression. Participants were 467 young adults , recruited from the general population. The subjects were also administered the Beck Depression Inventory-II (BDI-II). Results. Four first-order  and two higher-order factors  were extracted using Principal Axis Factoring analysis. Although all first-order and second-order factors were able to discriminate individuals with different depression severities, the Pessimism factor had the best performance in discriminating individuals with moderate to severe depression from those with lower depression severity. Conclusion. In the screening of young adults at risk of depression, clinicians have to pay particular attention to the expression of pessimism about the future. Major depressive disorder (MDD) is one of the most widespread psychiatric disorder –4 and thMany approaches have been taken in attempts to explain the origins of depression . Whereasnegative cognitive triad). Also, Carver and Ganellen [Despite similarities, currently there are several different theories which hypothesize the cognitive processes to be a diathesis for the development of depressive disorders. Two of the most renowned theories are the hopelessness theory  and BeckGanellen  considerSeveral researches have provided support for the critical role of CV in the origin and course of depressive disorders –30. For Our literature review suggests that investigating the role of CV in the development of depression may be beneficial for a better understanding and treatment of depressive disorders. What is more is that research has failed to investigate the role of CV for depression in discriminating different levels of severity of depression. Thus, the aim of the present study was to evaluate, by means of ROC curves, whether some dimensions of CV for depression, as measured by three well-known instruments, that are the Beck Hopelessness Scale (BHS) , the LifParticipants were 467 young adults . Mean age of the sample was 23.58 ± 5.03 years (range 18–39). Subjects were included if they had an age between 18 and 40 years old. Exclusion criteria were the presence of any condition affecting the ability to complete the assessment, including illiteracy and denial of informed consent. All participants were nonrandomly recruited in Central Italy between February 2012 and June 2012. They accepted to participate voluntarily and gave their informed consent.The Beck Depression Inventory-II (BDI-II) , the BHSThe BDI-II is a well-known self-report inventory composed of 21 items designed to assess the presence and severity of depressive symptoms, according to DSM-IV  criteriaThe BHS  is a 20-“I'm always optimistic about my future”), and the other three items are negatively worded . Each item is rated on a five-point Likert-type scale (from 1—“I agree a lot”—to 5—“I disagree a lot”). Sum scores range between 6 and 30 with higher scores indicating higher optimism.The LOT-R is a 6-item scale measuring optimism: three items are positively worded  to 5 (“I disagree a lot”). A study assessing the psychometric properties of the Italian version of the ATS-R has been recently published, indicating that the measure is a valid instrument for the study of the role of cognitive tendencies as potential diathesis in the development of depression [The ATS-R is a self-report measure designed to measure three potential self-regulatory vulnerabilities to depression. One of them is the holding of overly high standards, the second is the tendency to be self-critical at any failure to perform well, and the third is the tendency to generalize from a single failure to the broader sense of self-worth. The negative generalization scale has been found to be the dimension more strongly associated with depression , 47, 48.pression .The BHS, LOT-R, and ATS-R items were subjected to Principal Axis Factoring in order to obtain common factors of vulnerability for depression. The number of latent factors to retain was selected using the scree test  and confA generalized linear model with robust estimator was used to assess independent associations between depression severity and factors of vulnerability for depression. Associations were reported as odds ratio and their 95% confidence intervals (95% CI).In order to assess the performance of the dimensions of vulnerability for depression in categorizing individuals on the basis of depression severity, a series of ROC test procedures were performed .All analyses were performed with the Statistical Package for the Social Sciences (SPSS 17.0 for Windows).Descriptive statistics are listed in The scree test and Velicer's MAP test indicated a factorial solution with four factors, explaining 47% of the variability of the data . Nine itr = 0.20).Factor scores were entered in a second-order factor analysis to extract higher-order common factors. The analysis resulted in two factors with eigenvalues >1. On the first factor (Optimism), which explained 43.3% of the variance , first-order factors LOT-R Optimism (0.80) and BHS Optimism (0.72) loaded. On the second factor (Pessimism/Negative attitudes toward self), which explained 34.0% of the variance , first-order factors BHS Pessimism (0.62) and Generalized Self-Criticism (0.69) loaded. The factors were weakly correlated with each other . The four factors were inserted as independent variables in the analysis and the BDI-II as criterion. Only the BHS Pessimism and the LOT-R Optimism were independently associated with BDI-II scores, indicating that (1) subjects with higher scores on the BHS Pessimism were 52.4 times more likely to have higher BDI-II scores  compared with those reported having lower scores and (2) people with higher scores on the LOT-R Optimism were 9.1 times more likely to have higher BDI-II scores  compared with people having higher scores. A second generalized linear model was performed with higher order factors as independent variables (not reported in the tables). The model fitted the data well . Both the higher-order factors were independently associated with the BDI-II: (1) subjects with higher scores on Optimism were 10.1 times more likely to have higher BDI-II scores  compared with those having lower scores; and (2) people with higher scores on Pessimism/Negative Attitudes Toward Self were 91.1 times more likely to have higher BDI-II scores  than people reported having lower scores.A generalized linear model with robust estimator was performed to assess whether the factors measuring CV for depression were independently associated with the BDI-II . The modA series of ROC curves indicated that first-order and second-order factors of vulnerability for depression were able to discriminate individuals with different depression severities . The perThe aim of the present study was to evaluate, by means of ROC curves, whether CV for depression, as measured by three well-known instruments, may independently discriminate between subjects with different depression severities.Our results indicated that the instruments we administered measure four common vulnerabilities for depression: (1) denying optimism/endorsing high standards (BHS/ATS-R); (2) endorsing pessimism (BHS); (3) generalizing self-criticism (ATS-R); and (4) denying optimism (LOT-R). These dimensions are loaded on two second-order factors: (1) denying optimism and (2) endorsing pessimism and generalizing self-criticism.In our sample of young adults, the assessment of pessimism with the BHS, compared with other factors, had the best performance in discriminating individuals with different levels of depression severity, and its performance improved from lower cutoffs to higher cutoffs of depression. A randomly chosen individual with moderate to severe depression had 87% of probability to have higher BHS Pessimism compared with a randomly chosen individual with lower levels of depression. Pessimism was also independently associated with depression severity while controlling for other dimensions of vulnerability for depression. People having more severe pessimism were above 52 times more at risk to have higher depression compared with people with milder pessimism. This finding is consistent with the studies indicating a strong link between negative beliefs about the future and depression –58. For From a psychometric point of view, our results may be related to the differences in the methods employed in the three scales we administered to measure CV for depression: (1) the BHS uses dichotomous format of response, while for the LOT-R and the ATS-R the respondents are asked to rate each item on a Likert-type scale; (2) both the BHS and LOT-R ask the individuals to rate positive items which are directly associated with the construct they measure and negative statements which are negatively associated with these constructs. Furthermore, while the BHS is considered a measure of pessimism, the LOT-R is generally considered a measure of optimism . Scheier and Carver , investiNevertheless, in the last decades several authors reported results supporting the hypothesis that Optimism and Pessimism are two distinct constructs with different patterns of correlations with other psychological constructs –70. ReceFinally, our study has some limitations. First, above 60% of our sample were composed of university students and may not be representative of the Italian population, so that generalizability of the results to older people and to individuals with low school attainment is not suggested. Second, our sample is nonclinical and only 12.5% of the subjects reported having moderate to severe depression at the BDI-II, so that our results may not be replicable in clinical samples, even though cognitive theories of depression did not predict differences in the associations of cognitive variables with depressive symptoms between clinical and nonclinical samples , 72, 73.In conclusion, when screening young adults at risk for depression, clinicians have to pay particular attention to expressions of pessimism about the future, which may discriminate well individuals with moderate to severe depression from individuals reported having lower depression severity. Furthermore, at these levels of depression severity, Generalized Self-Criticism is less effective in discriminating individuals at risk for depression compared not only with statements of pessimism but also with statements denying optimism.


Recent evidence has shown that birch pollen-related soybean allergy mediated by Gly m 4 is common in central Europe. However, the impact of sensitization to Gly m 4 in soybean allergic patients in a population with a low prevalence of Betulaceae pollen allergy and a high soybean consumption is unknown.The aim of this study was to elucidate the prevalence of sensitization to rGly m 4 in adults with soybean allergy, and to analyze the diagnostic efficiency of the IgE antibody to rGly m 4 (ImmunoCAP®) in soybean allergy in central Japan. Twenty-one soybean-allergic patients were prospectively recruited from Jan. to Dec. 2009, and their levels of IgE antibody to rGly m 4 were compared with those of general alder pollen-allergic control subjects without soybean allergy (n=85).Although sensitization to alder pollen was not prevalent in the general outpatients of allergy departments, all the soybean–allergic patients were sensitized to alder pollen and rGly m 4. Sixty-two percent of the general alder pollen-allergic control subjects were also sensitized to rGly m 4. However, the levels of IgE antibody to rGly m 4 in soybean-allergic patients were markedly higher than those in alder pollen-allergic control subjects. The area under the receiver-operating characteristics curve for levels of IgE antibody to rGly m 4 in the diagnosis of soybean allergy was 0.86, which was significantly higher than that to the natural soybean extracts.A strong relationship between adult soybean allergy and sensitization to rGly m 4 was also observed in this population with high soybean consumption. The level of IgE antibody to CAP-rGly m 4 was an effective tool in discriminating soybean allergy from general alder pollen-allergy. This result highlights the impact of respiratory allergy to pollen-derived cross-reactive allergens on the epidemics of adult plant food allergy.


However, the potential contribution of TACE to skeletal development is still unclear. In the present study, we generated a Tace mutant mouse in which Tace is conditionally disrupted in chondrocytes under the control of the Col2a1 promoter. These mutant mice were fertile and viable but all exhibited long bones that were approximately 10% shorter compared to those of wild-type animals. Histological analyses revealed that Tace mutant mice exhibited a longer hypertrophic zone in the growth plate, and there were fewer osteoclasts at the chondro-osseous junction in the Tace mutant mice than in their wild-type littermates. Of note, we found an increase in osteoprotegerin transcripts and a reduction in Rankl and Mmp-13 transcripts in the TACE-deficient cartilage, indicating that dysregulation of these genes is causally related to the skeletal defects in the Tace mutant mice. Furthermore, we also found that phosphorylation of EGFR was significantly reduced in the cartilage tissue lacking TACE, and that suppression of EGFR signaling increases osteoprotegerin transcripts and reduces Rankl and Mmp-13 transcripts in primary chondrocytes. In accordance, chondrocyte-specific abrogation of Egfr in vivo resulted in skeletal defects nearly identical to those observed in the Tace mutant mice. Taken together, these data suggest that TACE-EGFR signaling in chondrocytes is involved in the turnover of the growth plate during postnatal development via the transcriptional regulation of osteoprotegerin, Rankl, and Mmp-13.TNFα-converting enzyme (TACE) is a membrane-bound proteolytic enzyme with essential roles in the functional regulation of TNFα and epidermal growth factor receptor (EGFR) ligands. Previous studies have demonstrated critical roles for TACE  Endochondral ossification is essential not only for the formation of cartilage models of future bones during embryogenesis but also for the longitudinal growth of the long bones during postnatal development. This process occurs in the growth plate  where resting chondrocytes differentiate sequentially into proliferative, pre-hypertrophic and hypertrophic chondrocytes. Histologically, the growth plate can be divided into four distinct layers: the resting zone, proliferating zone, maturing zone, and hypertrophic zone. In the hypertrophic zone, differentiated chondrocytes undergo apoptosis, and the cartilage matrix becomes calcified. The calcified matrix is subsequently invaded by newly formed vessels and absorbed by osteoclasts. Not surprisingly, many growth factors and transcription factors are involved in these processes, and the expression patterns of these genes are spatially and temporally regulated to achieve normal skeletal growth in vivo. However, the early lethality of TACE-deficient mice has hampered the analysis of TACE functions in postnatal development and adult homeostasis Tace was abolished in various tissues under the control of the Sox9 promoter exhibited defects in the hematopoietic system, skin, and fur, as well as severe growth retardation and low bone mass, indicating that TACE is involved in postnatal skeletal development TNFα-converting enzyme (TACE), also known as ADAM17 , is a membrane-bound proteolytic enzyme that is involved in the proteolytic release of the extracellular domain of the membrane-bound precursor of TNFα Tace was specifically disrupted in chondrocytes using Col2a1 promoter-driven cre-transgenic mice Mmp-13 transcripts, indicating that aberrant expression of these genes is casually related to the skeletal defects in Tace mutant mice. Furthermore, we found that phosphorylation of EGFR was significantly reduced in the cartilage tissues lacking TACE, and that inhibition of EGFR signaling in primary chondrocytes induces Opg transcripts, whereas suppresses those of Rankl and Mmp-13. In accordance, mutant mice with conditional ablation of Egfr in chondrocytes exhibited skeletal defects similar to those observed in the Tace mutant mice. Collectively, these data suggest that the TACE-EGFR axis plays a critical role in the turnover of calcified cartilage matrix through the regulation of Rankl, Opg, and Mmp-13 transcription, thus affecting osteoclast recruitment and matrix degradation.In the present study, we aimed to clarify the roles of TACE in postnatal skeletal development. We generated a mutant line in which flox/floxTace  mice with a transgenic line in which the transcription of cre was driven by a chondrocyte-specific Col2a1 promoter flox/flox/Col2a1-creTace , henceforth referred to as Tace/Col2 mice). As shown in Tace/Col2 newborn mice expressed very low levels of TACE protein. Tace/Col2 mice were fertile and viable. The offspring of Tace/Col2 and flox/floxTace  mice crosses were born in the expected Mendelian ratios (data not shown). Tace/Col2 mice did not exhibit overt skeletal defects or growth retardation (flox/floxTace counterparts (henceforth referred to as Control) at 8 weeks of age  and becomes less obvious as the mice reach maturity. On the other hand, there was no significant difference in the timing of vascular invasion into the secondary ossification center between Control and Tace-Col2 mice  labeling, but there was no significant difference in the number of BrdU-positive cells between the Control and Tace/Col2 mice (Control and Tace/Col2 mice (data not shown). In addition, an analysis of the femur revealed no difference in any of the histomorphometric parameters between the Control and Tace/Col2 mice, suggesting that bone homeostasis is maintained in Tace/Col2 adult animals . In contrast, there was no apparent difference in the distribution of osteoclasts on the secondary spongiosa (data not shown), which is consistent with the results of the histomorphometric analysis . To inactivate EGFR in chondrocytes, we also mated flox/floxEgfr mice with Col2a1-cre transgenic mice (flox/flox/Col2-creEgfr). As, shown in Tace/Col2 mice. Note that the elongation of the growth plate was not as significant as observed in Tace/Col2 mice, presumably due to an inefficient excision of Egfr alleles in flox/flox/Col2-creEgfr mice as previously described flox/flox/LysM-creEgfr mice .The present study shows that the chondrocyte-specific inactivation of TACE results in an elongated growth plate during postnatal long bone development. This defect is transient and becomes less obvious as the mice become older. By 8 weeks of age, the histological difference between Tace/Col2 mice compared to Control mice. Of note, we found that the Tace/Col2 cartilage exhibited lower Rankl and higher Opg transcripts compared with those in the control littermates, indicating that chondrocytes lacking TACE are defective in the recruitment of osteoclasts to the calcified cartilage. Because TACE is essential for the activation of several EGFR ligands, and phosphorylation of EGFR was suppressed in the Tace/Col2 cartilage, we examined how the inhibition of EGFR signaling would affect the expression of these genes. We found that treatment with a small-molecule EGFR inhibitor decreases the expression of Rankl and increases the expression of Opg in primary chondrocytes. Consistent with this observation, we also found that mutant mice lacking EGFR in the chondrocytes have skeletal defects similar to those in the Tace/Col2 mice. There are seven different EGFR ligands in mammals, and at least five of them  are highly dependent on TACE for their functional activation Tace-Col2 mice (shorter long bones and an expansion of the hypertrophic zone) Tace-col2 mice. In accordance, we also found that TGFα had a nearly opposite effect on the regulation of the transcription of Rankl and Opg in chondrocytes to those with EGFR inhibitor. Taken together, the present study suggests that EGFR signaling in chondrocytes positively regulates osteoclast recruitment, and that defective signaling in the TACE-TGFα-EGFR pathway in chondrocytes as the major cause for the phenotype of Tace-Col2 mice. On the other hand, because TACE has diverse target substrates, and the defect in the growth plate is transient, it is likely that compensatory or redundant mechanisms exist in osteoclast recruitment and degradation of calcified cartilage matrix at the chondro-osseous junction.Although TACE-deficient chondrocytes in the growth plate did not have apparent defects in cell proliferation or differentiation, there was a significant decrease in the number of TRAP-positive multinucleated osteoclasts adjacent to the chondro-osseous junction in the Tace/Col2 mice. Importantly, despite the differences in our animal models, our group and Zhang et al. −/−Egfr embryos exhibit delayed primary endochondral ossification due to defective osteoclast recruitment to the calcified cartilage While we were preparing the manuscript for the present study, another study describing the effects of gefitinib (Iressa) Mmp-13, and that Mmp-13 transcripts expression is reduced in the Tace/Col2 cartilage, indicating that elongation of the hypertrophic zone in Tace/Col2 mice was, at least to some extent, derived from a repression of MMP-13 activity. In all of these mutant mice, the elongation of the hypertrophic zone was caused by a defective degradation of calcified cartilage and not by developmental defects in the chondrocytes per se. Theoretically, delayed degradation of calcified matrix could also lead to a delay in the subsequent recruitment of osteoblasts and bone formation, and it is tempting to speculate that this delay in the turnover of the growth plate results in shorter long bones. Nevertheless, the exact cause-and-effect mechanisms between the elongated hypertrophic zone and shortened long bones observed in these mutant mice should be studied further.Similar defects in the growth plate  have been described in MMP-13-deficient In summary, we showed that the ablation of TACE in chondrocytes results in shorter long bones and elongation of the hypertrophic zone. Unexpectedly, there were no obvious defects in the differentiation or proliferation of TACE-deficient chondrocytes; however, the absorption of calcified cartilage was significantly delayed most likely due to the insufficient recruitment of osteoclasts to the chondro-osseous junction and reduced expression of MMP-13 . Althoug flox/floxTace mice, flox/floxEgfr  mice, Col2a1-cre transgenic mice, and LysM-cre transgenic mice has been described previously The generation of Tissue samples were fixed in 4% paraformaldehyde/PBS, paraffin-embedded, sectioned and then stained with hematoxylin and eosin or alcian blue solution. For BrdU labeling, BrdU (30 μg/g body weight) was intraperitoneally injected 3 hours prior to sacrificing the animal. Anti-MMP-9, anti-type-X collagen and anti-BrdU antibodies were obtained from R&D Systems, LSL, and Dako, respectively. The sections were imaged with a DS-Ri1 camera  on a BX53 microscope .Tace-siRNA (Dharmacon) introduced chondrocytes were incubated with serum-free medium for 24 h. At the end of the incubation, RNA was extracted using RNAiso  and reverse-transcribed using ReverTra Ace . PCR amplification and quantification were performed using SYBR premix EX Taq II (Takara Bio) and LightCycler II (Roche). The relative mRNA expression levels were obtained by normalizing the samples to the expression level of β-Actin transcripts. The sequences of the oligonucleotides used in this study are available upon request.Primary chondrocytes were harvested from newborn mice as previously described Tace/Col2 mice or control littermates were lysed in 1% TritonX-100 in PBS with 1 mM 1,10 phenanthroline, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma-Aldrich). The lysed cartilage tissues were further incubated with Concanavalin A sepharose beads  to purify the glycosylated proteins. The samples were separated by SDS-polyacrylamide gels and transferred to nitrocellulose membranes. TACE was detected using anti-sera against the cytoplasmic domain of TACE Primary chondrocytes, or cartilage tissues collected from the distal femur and proximal tibia from 7-days-old Control and Tace/Sox9 littermates. The femurs were excised, fixed with 75% ethanol, embedded in glycol methacrylate resin, and sectioned into 3 μm slices. For double labeling, the mice were subcutaneously injected with calcein (8 mg/kg body weight) at 1 and 4 days before sacrifice. The sections were stained with toluidine blue and subjected to histomorphometric analyses under a light microscope with a micrometer using a semiautomated image analyzer . The parameters for the trabecular bone were measured in an area of 1.62–2.34 mm2 at 1.2 mm above the growth plate at the distal metaphysis. Four mice from each group were analyzed.All of the histomorphometric analyses were performed in 8 week-old All of the experiments were conducted in triplicate. Standard deviations were calculated using Microsoft Excel. A student's t-test for two samples that assumed equal variances was used to calculate the P-values. P-values <0.05 were considered statistically significant.


In a biometric authentication system using protected templates, a pseudonymous identifier is the part of a protected template that can be directly compared. Each compared pair of pseudonymous identifiers results in a decision testing whether both identifiers are derived from the same biometric characteristic. Compared to an unprotected system, most existing biometric template protection methods cause to a certain extent degradation in biometric performance. Fusion is therefore a promising way to enhance the biometric performance in template-protected biometric systems. Compared to feature level fusion and score level fusion, decision level fusion has not only the least fusion complexity, but also the maximum interoperability across different biometric features, template protection and recognition algorithms, templates formats, and comparison score rules. However, performance improvement via decision level fusion is not obvious. It is influenced by both the dependency and the performance gap among the conducted tests for fusion. We investigate in this paper several fusion scenarios  on the binary decision level, and evaluate their biometric performance and fusion efficiency on a multi-sensor fingerprint database with 71,994 samples. However in this case, decryption is always required before comparison to tolerate the sample acquisition distortion of the fuzzy biometric signals. This demands an additional secure hardware environment for decryption and comparison. Moreover, standard encryption mechanisms become reversible when the secret key is available to the deciphering entity, such as in the system intrusion or insider attack scenarios. Unlike the standard encryption mechanisms which suffer from these drawbacks, many biometric template protection mechanisms have been proposed in recent years  achieves around 91% reduction in FRR compared to the best performance without fusion .Besides the above general observations, we found that only scenario (7) multi-sample-instance fusion and scenario (8) multi-sample-instance-algorithm fusion have some performance points that meet the recognition accuracy performance target for the project TURBINE. The best fusion result [FRR = 0.0060, FAR = 0.0009, while 5.2.Considering the number of decisions and convenience of finger probing can vary in different fusion scenarios, we need to evaluate the fusion efficiency in addition to the effectiveness we observed from the testing results and discussed in Section 5.1. To compare the fusion efficiency of different fusion scenarios in a more precise way than the general evaluation given in Section 5.1, we propose two criteria—efficiency per decision and efficiency per presentation—to evaluate the fusion efficiency.(1)i.e., exploiting the same amount of fusion sources. Since the number of decisions as fusion sources are equivalent, the performance is more comparable. E.g., the 2-sample fusion, 2-sensor fusion, 2-algorithm fusion, and 2 instances fusion cases are comparable since they have the same amount of source decisions. Following the same logic, other examples can be that the 4-sample fusion is comparable to the 2-sample-and-2-instance fusion, and the 3-sample-and-2-instance fusion is comparable to the 2-sample-and-3-algorithm fusion. By this efficiency per decision criterion, we can compare the different fusion cases' efficiency when they have the same amount of source decisions. Efficiency per decision is defined as the performance achievable by fusing the same amount of decisions, (2)Efficiency per presentation is defined as the performance achievable under the same amount of finger presentations (times of probing), which takes into account the convenience to the subjects. Since the effort required is almost the same under the same amount of finger presentations, the performance is more comparable. This efficiency criterion matters mainly on the multi-algorithm fusion since it needs only one finger presentation. 6.We presented in this paper an evaluation of decision level fusion results of fingerprint minutiae based pseudonymous identifiers generated by three biometric template protection algorithms developed in the European research project TURBINE. There are eight different fusion scenarios covering multiple samples, algorithms, sensors, instances, and their combinations in our tests. Distinct biometric performance improvements were observed by decision level fusion which verifies the hypothesis to apply fusion to improve the recognition accuracy performance. For fair comparisons of achieved performance improvement, two fusion efficiency criteria were proposed to evaluate the different scenarios' fusion efficiency.Future work will investigate other fusion rules such as AND, layered, and cascaded fusion; and also the in-depth implications of performance improvement by the fusion rule OR and the dependency among elements for fusion. With regards to privacy, we assume that the template protection algorithms used are secure and privacy-enhanced. However, decision level fusion implies linkability among the protected templates generated out of different samples, sensors, instances, and algorithms from the same biometric characteristic  and the same subject (in the instance case). Whether or not this fact influences the protected templates' security is quite algorithm-dependent, and therefore needs case-based security analysis.