To analyze speech reading through Internet video calls by profoundly hearing-impaired individuals and cochlear implant (CI) users.Speech reading skills of 14 deaf adults and 21 CI users were assessed using the Hochmair Schulz Moser (HSM) sentence test. We presented video simulations using different video resolutions , frame rates ), speech velocities (three different speakers), webcameras  and image/sound delays (0–500 ms). All video simulations were presented with and without sound and in two screen sizes. Additionally, scores for live Skype™ video connection and live face-to-face communication were assessed.Higher frame rate (>7 fps), higher camera resolution (>640×480 px) and shorter picture/sound delay (<100 ms) were associated with increased speech perception scores. Scores were strongly dependent on the speaker but were not influenced by physical properties of the camera optics or the full screen mode. There is a significant median gain of +8.5%pts (p = 0.009) in speech perception for all 21 CI-users if visual cues are additionally shown. CI users with poor open set speech perception scores (n = 11) showed the greatest benefit under combined audio-visual presentation .Webcameras have the potential to improve telecommunication of hearing-impaired individuals. For many years the use of videophones for transmission of sign language or lip motion over telephone networks was either expensive or of low image quality, thereby limiting its use Video telephony as provided by Skype™ and other Internet communication companies offer a broadband transmission of voice and image over an Internet protocol (IP) network. The Internet software sends small packets of encoded data over the Internet guided via the IP. Each data packet takes a unique pathway through the network before arriving at a receiver computer that uses the same software as the sender. The receiver’s software then collects, reconstructs and decodes all data packets before finally converting them back into an analog signal that is presented to the end-user.Despite its potential benefits, the quality of internet video telephony transmission may be hampered by congested internet lines The study protocol was approved by the local institutional review board ; all patients gave written informed consent.All tests were conducted between March 2010 and July 2011 at the Department of Otorhinolaryngology-Head and Neck Surgery, Inselspital, University of Bern, Switzerland. In total, 14 deaf adults and 21 CI users participated in the study. We chose deaf individuals as a reference for the assessment of speech reading and CI users for the assessment of both speech reading and audiovisual gain. Deaf individuals were recruited from deaf community organizations . Eight individuals had congenital deafness , four had prelingual deafness  and two individuals lost their hearing at the age of 22 and 44 y (progressive hearing loss). The mean age of this group was 41.6 years (range 23–63 years). All 14 deaf adults used sign language and speech reading in daily communication. They had normal vision or normal corrected vision.CI users were recruited through a database of the audiological department of our tertiary referral center. The mean age of CI users at time of cochlear implantation was 31.3 years (range 3–63 years) and 40.6 years at the time of testing (range 18–70 years). All CI users were therefore experienced, with a mean CI-listening experience of 9.3 years (range 4–22 years). All 21 CI users had bilateral profound hearing loss, normal vision or normal corrected vision, and used an auditory-oral or total communication mode in daily life. We performed three series of experiments to assess speech reading cues transmitted over the Internet. A first set of experiments assessed speech reading in deaf adults under controlled conditions. Factors hypothesized to influence Internet speech communication such as different speech velocities (different speakers), camera properties , screen properties (resolution) and Internet transmission rates (resulting in a specific frame per second [fps]) were tested by a video simulation. We presented video simulations using three different speakers , four screen resolutions , two screen sizes , five frame rates , and three web cameras . Details about the digital generation of audio-visual video files for the simulation are shown in A second set of experiments tested speech reading skills in deaf individuals and CI users under real but controlled conditions  by using a Skype™ Internet connection (250 kBps download and 3 kBps upload speed) between two rooms. A telephonometric communication (two persons communicate in the same room at 1 m distance) served as a reference standard. A third set of experiments on CI users aimed to assess audiovisual cues transmitted by Internet video telephony. Auditory and combined AV stimuli with different AV-delays (0–500 ms) were used under simulated and real network conditions. Patients were asked about their overall experience with AV delay and whether they felt they required both auditory and visual stimuli or relied on either auditory or visual stimuli alone.The German “HSM” sentence test All CI users were tested monaurally using the same ear throughout the entire test battery. Bilateral CI users were asked to remove the device on the poorer hearing side in order to obtain more homogenous data across subjects. The opposite ear canal was occluded with an ear plug  if residual low-frequency hearing was present. The specified average attenuation of these earplugs is 24.6 to 41.6 dB in the range of 250 to 4000 Hz. For the live speech reading test (Skype™ versus face-to-face) without acoustical input , CI users were required to power off their devices and to use ear plugs bilaterally.Testing was performed in a single session with a total testing time per subject of approximately 2 hours including breaks.Bonf).Robust nonparametric analyses were performed to assess the potentially non-normally distributed speech perception scores from this small study population. We used the Spearman correlation test to assess the relationship between speech perception and camera properties (frame rate and resolution [megapixel]). To evaluate speech reading performance, we first used the Friedman test to identify possible differences at a 0.05 significance level in each of the following parameters: camera types, communication modes, speakers and AV-modes. Then, we used the two-tailed Wilcoxon matched pairs signed-rank test to compare groups within the parameters that were identified as different from the Friedman test. Bonferroni correction was performed for multiple testing (PAll 14 deaf adults had measurable speech reading abilities (face-to-face without sign language) with speech perception scores ranging from 41.5%–97.2% (median 74.5%). When video files were presented, scores were lower and ranged from 1.9% to 75.5% (median 51.9%), depending on the speaker. The slower speaking individual ‘CD’ (97 words/s) was better understood and this resulted in substantially higher scores (median 52.4%) compared to presentations of files by the other two speakers . The score differences from speaker ‘CD’ to ‘JB’ and ‘SF’ were statistically significant .Similarly, the communication mode influenced speech perception by deaf individuals as shown in The type of camera hardware did not greatly influence speech reading scores by deaf individuals; none of the comparisons across camera types reached statistical significance , however, deaf individuals showed greater degradation of speech reading performance during a Skype™ video call (median 50.9% for npCI versus 35.9% for deaf subjects).Audio-visual asynchrony was associated with lower speech perception scores by CI users. The association between duration of the AV-delay and the speech perception scores are directly correlated for short delays . To our knowledge, this is the first study investigating speech perception using Internet video telephony technology available on the market.Speech reading is often used in conjunction with sign language or with an auditory input (AV-mode) if applicable. A multimodal communication mode increases speech perception performance by deaf and CI-using individuals. Audio-visual speech perception depends on several factors such as speech reading abilities, auditory performance and capacity of AV-fusion. CI users rely on bimodal speech comprehension, especially for face-to-face communication under noisy conditions where the auditory speech signal is degraded. Speech reading is still possible after cochlear implantation and does not change within the first postoperative year with recruiting of visual and audio-visual brain areas during communication Together with directional microphones, AV cues improve speech perception performance under adverse listening conditions Furthermore, the transmission of speech reading cues over a video screen or via Skype™ transmission led to lower speech perception scores in comparison to a face-to-face communication mode  and 3. TFoulds et al. It has been reported that communication by speech reading and sign language at a resolution of 176×144 pixels is possible despite losing many facial details According to Luca De Cicco et al End-to-end video delay should be kept below 0.4 s AV signals are often synchronized by a form of interlaced video and audio data or by explicit AV synchronization by time-stamping AV asynchrony may be related to calculation delay in the cochlear implant system, however we did not test this. Based on manufacturer data, the processing time in the implant or speech processor is negligible compared to the AV-delays occurring in video telephony.One limitation of the presented study is the fact that only speech reading based speech perception by deaf individuals was assessed. The effects of sign language or bimodal communication (lip movements and sign language combined) on speech perception through video-transmission have not been considered. The main reason for focusing on speech reading was that all participants  had some experience reading lip reading cues. Not all tested CI users were able to understand sign language. In addition, this study aimed to understand the potential of current technology for transmitting lip motion cues over the network, which is more delicate compared to hand and finger motion cues Internet video telephony (in particular Skype™) offers direct communication benefit for deaf and cochlear-implanted individuals at minimal cost. We believe the four main advantages of this new technology for CI users are: 1. Bilateral hearing is possible either in free field with PC active loudspeakers or with headphones, 2. the auditory signal can be amplified up to a comfort level, while the conventional telephone is adjustable only to a limited extent, 3. broadband voice quality is near CD-quality in comparison to a low-pass filtered signal in conventional telephony The present study identified several factors associated with improved speech reading performance over Internet video telephony, such as frame rates above 15 fps, camera resolution above 640×480 px, slower speaker and shorter audio-visual delay (<100 ms). Overall, Internet video telephony transmits sufficient lip shape information for speech reading by deaf and cochlear-implanted individuals. There are significant audio-visual benefits observed for CI users; however, bimodal cues with the addition of sign language for deaf individuals or auditory input for cochlear-implanted patients are still recommended for engaging in meaningful video-conversation over the web.Text S1Digital generation of audio-visual video files.(DOC)Click here for additional data file.Text S2Live Skype™ transmission.(DOCX)Click here for additional data file.


Non-melanoma skin cancers are one of the most common human malignancies accounting for 2–3% of tumors in the US and represent a significant health burden. Epidemiology studies have implicated Tp53 mutations triggered by UV exposure, and human papilloma virus (HPV) infection to be significant causes of non-melanoma skin cancer. However, the relationship between Tp53 and cutaneous HPV infection is not well understood in skin cancers. In this study we assessed the association of HPV infection and Tp53 polymorphisms and mutations in lesional specimens with squamous cell carcinomas.We studied 55 cases of histologically confirmed cutaneous squamous cell carcinoma and 41 controls for the presence of HPV infection and Tp53 genotype (mutations and polymorphism).We found an increased number of Tp53 mutations in the squamous cell carcinoma samples compared with perilesional or control samples. There was increased frequency of homozygous Tp53-72R polymorphism in cases with squamous cell carcinomas, while the Tp53-72P allele (Tp53-72R/P and Tp53-72P/P) was more frequent in normal control samples. Carcinoma samples positive for HPV showed a decreased frequency of Tp53 mutations compared to those without HPV infection. In addition, carcinoma samples with a Tp53-72P allele showed an increased incidence of Tp53 mutations in comparison carcinomas samples homozygous for Tp53-72R.These studies suggest there are two separate pathways (HPV infection and Tp53 mutation) leading to cutaneous squamous cell carcinomas stratified by the Tp53 codon-72 polymorphism. The presence of a Tp53-72P allele is protective against cutaneous squamous cell carcinoma, and carcinoma specimens with Tp53-72P are more likely to have Tp53 mutations. In contrast Tp53-72R is a significant risk factor for cutaneous squamous cell carcinoma and is frequently associated with HPV infection instead of Tp53 mutations. Heterozygosity for Tp53-72R/P is protective against squamous cell carcinomas, possibly reflecting a requirement for both HPV infection and Tp53 mutations. Tp53 gene mutations, characteristically (C to T or CC to TT) transitions at dipyrimidine sites Non-melanoma skin cancer (NMSC) is now the most common cancer among Caucasians, outnumbering the total of all other cancers combined Over the last 10 years there has also been increasing interest in the potential role of cutaneous human papillomavirus (HPV) in the development of SCC. However, whether certain HPVs play a role in development of skin SCC, and if so, how, is not well understood. Data supporting a role for cutaneous HPV in the development of skin SCC include a high rate of malignant transformation (i.e. development of SCC) of sun-exposed skin warts among subjects with either inherited immunosuppression (such as patients with epidermodysplasia verruciformis (EV), who lack the ability to control infection with specific cutaneous HPVs Laboratory based studies suggest that certain Tp53 polymorphisms may also play a role in the pathogenesis of NMSC. A common Tp53 polymorphism located at codon 72 (encoding either a proline or arginine residue), within a conserved proline rich SH3 binding domain, is known to be critical for promoting apoptosis While previous studies have examined the relationships between NMSC and HPV infection, or NMSC and UV-induced Tp53 mutations and polymorphisms, to our knowledge, none have examined all these factors in one study. In the present study, we examined the relationship between HPV infection, Tp53 mutations and Tp53 codon 72 polymorphisms in tissue biopsies of tumor and surrounding histologically normal skin from patients with NMSC, and from sun exposed and non-sun exposed tissue of individuals without NMSC. We hypothesize that several different pathways lead to the development of NMSC.The inclusion and exclusion criteria, subject recruitment, interview procedures, physical examination, and collection of specimens have been previously described The present study population consisted of 55 patients (cases) and 41 controls on whom detection and sequencing of Tp53 mutations was conducted. Of the 96 study subjects, 48 (18 cases and 30 controls) were included in our previous report regarding HPV and SCC For cases, a portion of the residual SCC along with adjacent clinically uninvolved perilesional skin was collected from each subject. For controls, punch biopsies of clinically normal skin were obtained from a sun-exposed (pre-auricular) site as well as from a sun-protected site from the upper inner arm as previously described. One third of the biopsy was placed in 10% neutral buffered formalin for histopathology, another third placed into specimen transport media , and the final portion placed into ethanol.Routine paraffin-embedded tissue was prepared from the biopsy specimens and then sectioned and stained with hematoxylin and eosin to confirm the diagnosis. HPV was detected by PCR and typed as previously described using three protocols to assure detection of all HPV types Since our goal was to identify major Tp53 mutations that are likely involved in the pathogenesis of neoplasia, we focused on identifying clonal mutations (most abundant in neoplastic or pre neoplastic tissue), rather than sub-clonal mutations (present at lower frequencies). To this end we directly sequenced coding sequence (cDNA) amplified from tissue specimens without employing a clonal enrichment steps Tp53 sequence was evaluated for the presence of either non-synonymous , synonymous (no change in amino acid sequence) mutations and UV damage signature  Differences between cases and controls were assessed using chi-square analyses to compare categorical variables and Student’s t-test to compare continuous measures. Associations with ordinal factors, such as propensity to sunburn, were evaluated with Mantel-Haenszel tests for trends. Fisher’s exact tests were utilized when cell frequencies were less than 5. To evaluate differences in categorical measures by site of the skin tissue, paired samples from within a subject were compared using McNemar’s Test.A two-sided 0.05 level test determined statistical significance for all analyses. All analyses were conducted using SAS 9.1 .The characteristics of the study population including 55 subjects with and 41 without cutaneous SCC are presented in Among cases, the frequency of any Tp53 mutation was significantly increased in the lesional samples (33%) as compared to perilesional samples (16%). However, Tp53 mutations were rare in both sun exposed (2%) and sun protected (5%) control tissues . IdentifCharacterization of the specific type of Tp53 mutations showed that 17 of 18 lesional tissues with Tp53 mutations were non-synonymous mutations (resulting in amino acid changes), and 13 of these had mutations with a UV damage signature . 8 of 9 Tp53 mutations from perilesional tissue were non-synonymous, with 7 exhibiting a UV damage signature. The single Tp53 mutation identified in the sun-exposed control was synonymous (no amino acid change), while the two mutations identified in the sun-protected controls were non-synonymous mutations with a UV damage signature. Among tumor tissues with Tp53 mutations, 11 of 18 (61%) were previously reported “hotspot” mutations, while among perilesional tissues 4 of 9 (44%) were “hotspot” mutations. Surprisingly only 1 of 5 (20%) of the lesional and perilesional tissues showed concordant Tp53 mutations.Since, as mentioned above some In the present study, 66% of the lesional samples, 55% of the perilesional samples, 60% of the sun-exposed control samples, and 51% of the sun-protected control samples had detectable HPV DNA. Although we previously reported that HPV β-papillomavirus species was differentially detected in lesional (tumor) as compared to perilesional tissue in the larger sample set, there was no difference between lesional, perilesional and control tissues with respect to the specific types of HPV present in this smaller sample set. Interestingly, detection of HPV was inversely associated with the presence of Tp53 mutations among lesion tissues . While TGiven the above findings, we asked whether the role of Tp53 mutations and of HPV infection in the pathogenesis of SCC might vary with a specific polymorphism at Tp53-codon 72 . Among lNon-melanoma skin cancers are frequent tumors that arise from a complex interaction between environmental, immunologic and genetic factors As expected there was an increase in the frequency of Tp53 mutations in the tumors compared with either the perilesional or control specimens. Most of the Tp53 mutations present in the cancers were non-synonymous changes and many were previously characterized “hotspots” that showed signatures of UV damage suggesting exposure to sunlight is a primary environmental insult causing the mutations For this study we were interested in identifying the most prevalent p53 mutations that might represent driver mutations promoting SCC. Therefore we choose to use direct cDNA sequencing without a clonal enrichment step such as denaturing high-performance liquid chromatography (DHPLC). This procedure allows for greater specificity in identifying clinically relevant mutations that promote carcinogenesis. However this approach does not detect every p53 mutation present in the sample since small subclonal mutations would not be identified without a clonal enrichment step. In addition, our sequencing strategy targeted exon 4 to exon 8, spanning the region that encodes most of the allelic polymorphisms and reported mutations. P53 mutations occurring outside of this region would not be detected in this study. Predominant mutations in Tp53 were readily identified in tumors representing a clonal population of cells sharing identical Tp53 mutations. Presumably, p53 sequences amplified from control and perilesional samples were heterogeneous and contained subclonal Tp53 mutations amid a background of wild-type Tp53 sequences; minor Tp53 mutations were not detected by direct sequencing. The frequency of Tp53 mutations detected in the perilesional and control specimens were lower than that reported in other studies focusing on squamous cell carcinomas that employed a clonal enrichment procedure In a recent study Durinck et al. determined the temporal sequence of acquired genetic abnormalities during tumor progression by using a combination of DNA sequence and copy number variations Of the 56 paired samples  only 5 had Tp53 mutations identified in both tissues and only 1 (20%) shared the same mutation. The lack of tumor-specific p53 mutations in the adjacent perilesional is probably due to sampling bias and the limited sensitivity of direct cDNA sequencing. Ren et al. found that benign clonal keratinocyte patches with p53 mutations showed no genetic correlation to adjacent squamous cell carcinomas. These authors suggest the p53 expressing keratinocyte patches result form clonal expansion of cells with p53 mutations that may be permissive for a subsequent event driving carcinogenesis. In the vast majority of cases these p53 mutations do not represent precursor mutations present in the adjacent carcinomas Although direct causal association between HPV and cervical carcinoma has been well established in vitro studies have shown that expression of wt Tp53 can inhibit HPV replication by blocking the expression, and promoting the degradation of HPV20 E6 We hypothesized that HPV infection and Tp53 mutations would be increased in carcinoma samples with a positive and cooperative association in promoting carcinomas. Instead, we found an inverse association; HPV positive tumors had a lower incidence of Tp53 mutations (23%) in comparison to HPV negative tumors (50%). This inverse association was not present in perilesional or control samples suggesting that this relationship pertains to the induction of tumors. The simplest explanation for these results is that two potential separate pathways either dependent on HPV infection or on Tp53 mutation lead to cutaneous squamous cell carcinoma. HPV infection may decrease the incidence or necessity of Tp53 mutations, and Tp53 mutations may inhibit HPV infection or the proliferation of HPV infected cells. A similar inverse correlation has been reported in oropharyngeal squamous cell carcinomas Although the E6 and E7 proteins encoded by cutaneous HPVs do not bind or target Tp53 or RB protein for degradation By stratifying patients based on the status of the common Tp53 polymorphism at codon-72(R/P) we identified several additional statistically significant associations that lead to important mechanistic predictions. The lack of Tp53-72P (Tp53-72R/R) was a significant risk factor for SCC in the study population of elderly white male patients. Tumors without Tp53-72P (Tp53-72R/R) had fewer Tp53 mutations with a small non-significant increase in HPV DNA. These results suggest that patients homozygous for Tp53-72R are more likely to develop cutaneous squamous cell carcinoma with HPV infection and without Tp53 mutations. These results resemble those reported for SCC arising in immunosuppressed patients. It is well documented that there is an increased incidence of skin cancers in selective immunosuppressed patient populations (solid organ transplant and epidermodysplasia verruciformis (EV)) attributed to potential infectious or immune-modulatory factors The codon-72 polymorphism is located in a highly conserved proline rich domain involved in DNA binding, and several studies show clear functional differences between the two isoforms. Expression of the Tp53-72R in p53-null cells induced enhanced apoptosis possibly related to increased localization of the arginine variant to the mitochondria compared to the proline variant Similar to the cell cycle studies the association of the common Tp53 polymorphisms at codon-72 with cancer risk has been extensively studied for multiple cancers with mixed and inconsistent results. There are over 230 studies in the NIH genetic association database evaluating the effect of the codon-72 polymorphism on susceptibility to a wide variety of cancers with many reporting ‘statistically significant’ associations Among the carcinomas without detectable Tp53 mutations there was a non- significant trend toward an increased frequency of HPV infection in tumors lacking Tp53-72P . This trOur results show that tumors expressing the Tp53-72P polymorphism showed an increased frequency of Tp53 mutations (55%) in comparison to tumors without Tp53-72P (20%) consistent with studies in lung carcinoma In a recent study, Guan et al. showed that ARID1a, a tumor suppressor gene involved in chromatin remodeling and frequently mutated in a wide variety of tumors binds to p53 In conclusion, our studies demonstrate a clear association between Tp53 mutations and squamous cell carcinomas. The association with HPV infection is less well defined partly due to the high incidence of HPV infection in normal and tumor biopsies. It is possible that specific HPV types are causally linked with cutaneous squamous cell carcinomas and were not identified in this study since all HPV infections were classified together. Studies monitoring other aspects of HPV infection including viral load, integration and expression may better define a causal link with cutaneous squamous cell carcinoma. Importantly we found that the expression of the common Tp53 polymorphism Tp53-72P is protective against the development of cutaneous squamous cell carcinomas arising in elderly white males. In addition, we found a significant inverse correlation between Tp53 mutations and HPV DNA in the tumors stratified for Tp53-72 polymorphism suggesting two separate mechanisms of carcinogenesis one dependent on Tp53 mutations and the other dependent on HPV infection.Table S1Primers for Tp53 analysis. The Tp53 mRNA template was transcribed into cDNA with MLV reverse transcriptase, using the listed cDNA amplification primers (1) to generate a 1142 bp product. Nested PCR reactions were carried out to amplify an 873 bp fragment from exon 4 to exon 8 using the nested PCR amplification primers (2). Both strands of the amplified cDNA were then sequenced using the specific sequencing primers (3).(DOCX)Click here for additional data file.


Clinically, at a 9-year average followup, both groups had comparable HHS scores and incidence of thigh or groin pain, though the machined group had an increased incidence of osteolysis and annual linear wear rate.Multiple factors have been identified as contributing to polyethylene wear and debris generation of the acetabular lining. Polyethylene wear is the primary limiting factor in the functional behavior and consequent longevity of a total hip arthroplasty (THA). This retrospective study reviewed the clinical and radiographic data of 77 consecutive THAs comparing in vivo polyethylene wear of two similar acetabular cup liners. Minimum follow-up was 7 years (range 7–15). The incidence of measurable wear in a group of machined liners sterilized with ethylene oxide and composed of GUR 1050 stock resin was significantly higher (61%) than the compression-molded, GUR 1020, O Ultra high molecular weight polyethylene (UHMWPE) is currently the most widely used polymer for joint replacement prosthesis. Polyethylene mm head were used in all cases. The ceramicwear is the primary limiting factor in the functional behavior and consequent longevity of a total hip arthroplasty (THA). Polyethylene debris has been often linked to the development of osteolysis with subsequent loss of bone stock and implant fixation . Linear Multiple factors have been identified as contributing to polyethylene wear and debris generation of acetabular lining. These variables include conformity of the articulating surface, polyethylene thickness, femoral head diameter, polyethylene locking mechanism, polyethylene additives , sterili2-free gamma irradiation. This grouping of polyethylene liners will henceforth be referred to as Group 2. The two hip systems had similar polyethylene locking mechanisms, articulating head surfaces, and articulating head geometry. The acetabular polyethylene liners of one of the total hip systems were machine fabricated using a bar extrusion technique from GUR 1050 stock resin, before being sterilized with ethylene oxide (EtO). This group will henceforth be referred to as Group 1. The other hip design uses compression molded polyethylene, from GUR 1020, resin and was sterilized using OIn the past decade, there has been significant progress in the development of a more wear and oxidation resistant UHMWPE alternative such as the highly cross-linked UHMWPE. Initial lab and clinical studies have shown that cross-linked polyethylene may be more wear resistant than non cross-linked alternatives . The focInstitutional Review Board approval was granted for this study. Criteria for this study included THA cases with at least 7-year followup, with standard polyethylene (with a minimum of 7 mm thickness), with the same diameter femoral heads 28 mm Alumina Ceramic (Al203) or Cobalt Chrome (CoCr), and one of two press fit titanium acetabular cups. The CoCr femoral heads of both groups were from the same manufacturer. Seventy-seven THAs, performed by the same surgeon that occurred between February 1996 and August 2003, with these criteria were retrospectively reviewed. Clinical and radiographic data-collected including presurgery diagnosis, patient demographics, pre- and postsurgery Harris Hip Scores (HHS), incidence and severity of polywear, osteolysis, and patient reporting thigh and/or groin pain. Refer to This study compares clinical and radiographic assessment of polyethylene wear of two different acetabular cup liners, Group 1  and Group 2 . Each liner studied articulates with either a cobalt chrome femoral head  or a ceramic femoral head (Alumina)  and has effective and similar locking mechanisms within the acetabular cup to minimize backside wear.2O3 28 mm head were used in all cases. The ceramic heads were used primarily in younger male patients with physically demanding jobs. Postoperative rehabilitation included patients being weight-bearing as tolerated with walker support for 3–6 weeks as needed. Venous thrombosis event (VTE) prophylaxis was based on an individual patient VTE risk assessment. For the standard at risk patient included oral warfarin 5 mg the night of surgery and continued daily until the ProTime (prothrombin time) reached 15 seconds or the INR  reached 1.2–2. Oral warfarin 2 mg was then given daily for a 4-week protocol of 2 mg per day mini-fixed dose oral warfarin regimen. For the patient without higher VTE or bleeding risk assessment, once the hospital ProTime reached 15 seconds or INR  levels reached 1.2–2.0, post discharge monitoring was not done unless signs or symptoms of bleeding occurred. For higher risk VTE patients, higher dose monitored oral warfarin was used (ProTime of 18–20 seconds or INR range 2.0–2.5). All patients were counseled to avoid dislocation-prone lower extremity positioning of surgical leg internal rotation and adduction and maintain less than 90 hip flexion for 12 weeks following surgery.All surgeries were performed using a posterolateral approach. Thirty-one press fit Smith and Nephew Reflection acetabular cups (Group 1) and 46 press fit Zimmer Intraop acetabular cups (Group 2) were implanted. Cup screws were used based on surgical indication for cup fixation and stability. The natural hip press fit stem and a corresponding CoCr or AlRadiographic assessment of osteolysis was performed in each case at the most recent annual clinical follow up-period and classified based on Gruen et al.'s and DeLee and Charnley's classification for the femoral stem and acetabulum respectively , 14.α = 0.05).Linear polywear rate and cup abduction/inclination angle were measured on digital radiographs using femoral head size to standardize magnification. Linear wear was assessed with a resolution of 0.1 mm using techniques described and validated by Griffith et al. and Livermore et al. , 16. LinBoth titanium cup designs incorporated secure polyethylene inserts with locking mechanisms that significantly restrict both rotational and pistoning motions to minimize backside wear potential. Both cups incorporate hemispherical geometry with roughened outer surfaces for acetabular bone ingrowth. All polyethylene liners were a minimum of 7 mm actual thickness with ranges from 7 to 15.5 mm depending on the size of the individual patient's acetabulum and subsequent cup diameter. The acetabular cups liners differ primarily with respect to stock resin, manufacturing methods, and sterilization techniques .  FigureSEM images were taken using a variable pressure Hitachi 3400 SEM at 20 keV accelerating potential capability at magnification of 40x and 95x. It is equipped with Phillips Secondary Electron Detector 6765/50 complimented by a Hitachis Backscattered Electron.t-test with α = 0.05) was performed to evaluate whether there was a significant difference between machined and compression molded nonarticulating liner surface roughness. The effect of the manufacturing procedure on the surface of the UHMWPE liners (machined and compression molded) was evaluated using noncontact profilometry using a WYKO NT2000 profilometer . Surface characterization was performed after retrieval at a nominal magnification of 25x . To best capture the manufactured original surface, Nonarticulating surfaces were characterized for roughness measures arithmetic surface roughness (Ra), average maximum profile peak height (Rpm), and average maximum profile valley height (Rvm). Five measurements were taken on the nonarticulating surface of each polyethylene liner in a linear fashion in order to fully quantify and characterize the component and ensure reliable and repeatable estimate of nonarticulating surface roughness. Statistical analysis  with lenses providing 6x to 50x magnification with controlled fluorescent ring illumination and a color digital camera .P = 0.0004). The linear wear rate of Group 1 was additionally significantly higher (2.7 times) than Group 2 polyethylene's with CoCr head group (P = 0.0014). The linear wear rate of Group 1 was also significantly higher (1.8 times) than the Al2O3 Group 2 (P = 0.0028). There was no difference in the wear rate between the two subgroups in Group 2 when comparing those with CoCr heads and those with Al2O3 (P = 0.071)  was significantly higher than that of Group 2 (23.9%)  .Nakahara et al. have shown that acetabular cups implanted with >45° abduction have higher polyethylene wear rates than cups implanted with 45° or less . When asThe complications in each group were comparable and no statistical difference was found in the complications in either group refer to .This study examines the clinical polyethylene wear characteristics of two independent total hip systems. Upon comparison, significant statistical differences are found between the polyethylene wear seen within the two systems. The polyethylene of Group 1, made of GUR 1050 resin, sterilized through the use of ethylene oxide, was manufactured by ram extrusion and machining. Group 2 polyethylene is compression-molded from GUR 1020 resin and is sterilized by means of gamma irradiation in an oxygen free-environment. In vitro studies on tibial inserts for knee arthroplasties have shown that compression molded and oxygen free sterilization reports less wear than machined tibial inserts for total knee arthroplasties , 17, 18.2 has shown to increase polyethylene oxidation due to free radical generation and subsequent oxidation, consequently lowering mechanical properties, leading to rapid polyethylene wear. However, gamma irradiation performed in an oxygen free environment has been shown to negate the oxidative effects of irradiation [The polyethylene of Group 2 was sterilized via oxygen-free gamma irradiation. Gamma irradiation in the presence of Oadiation . Faris eadiation .Each liner was also formed from two different polyethylene resins, GUR 1050 for Group 1 and GUR 1020 for Group 2. The lone difference between the two resins is the average molecular weight, which is around 3.5 × 106 g/mol for GUR 1020 and 5.5 × 106 for GUR 1050. Past studies have shown that the higher molecular weight provides better abrasive wear resistance, but only slightly. Kurtz  argued t2O3 with respect to polyethylene clinical wear rate in THAs [2O3 the range is 0.03–0.15 mm/year [2O3 head versus CoCr heads, though the linear wear rates reported in this study fall within the expected range. This may be explained by the practice of using Al2O3 heads in younger more active patients. While there was not a difference in average age or range, there was a tendency for more males in the compression molded Al2O3 head group. There was a trend towards younger males in the Al2O3 head group compared to either group (machined or compression molded) with CoCr. This was not statistically different (Previous authors have compared the bearing material effect on CoCr versus Al in THAs . Clinica mm/year , 21. Whiifferent .The final variable of interest that could significantly affect polyethylene wear is the manufacturing technique. There are various types of fabrication methods that can be employed in the fabrication of polyethylene orthopedic implants. Most of the acetabular liners used today are machined from extruded bar stock or compression-molded directly from the polyethylene powder resin . Machineμm for machined and 0.40 μm for molded surfaces, which are characteristic of these surfaces in UHMWPE implants [In addition to the maldistribution of solid-phase polyethylene within the ram extruded bar stock, surface characteristics and topography may have a significant role in polyethylene oxidation. In the machined polyethylene insert, machine marks from the lathe creates numerous micron size grooves and shreds on the bearing surface, which are not found on the surface of the compression molded polyethylene insert. This was confirmed in the current study as shown in implants . DespiteIn summary, the implant design using GUR1050 bar stock, sterilized in ETO, with final articular surface geometry machined had significantly more linear wear and radiographic osteolysis. The implant using GUR 1020 stock powder, compression-molded into final surface geometry without machining, and sterilized in inert gamma irradiation showed significantly less radiographic measured wear and osteolysis. It is beyond the scope of this paper to conjecture which of the differing elements is responsible for the greater linear wear rate in one implant versus the other. PE wear rates have been shown to predict osteolysis, implant longevity, and revision surgery, and it is routine practice to monitor all implants for wear and signs of failure. Most current THA implants are using highly cross-linked PE because of more favorable reported wear rates. However, there are a large number of hip arthroplasty implants using conventional PE that were implanted before highly cross-linked PE was available. Cross-linked PE has also been slow to move into wide-scale implementation on a global scale due to its high cost and low availability outside of the United States. Thus, understanding the fundamental core materials and the manufacturing process for conventional PE implants and having comparative clinical reviews as described here suggest that it may be prudent to monitor some implants more closely than others for polyethylene wear-related signs of problems.


V2-type channels. Increased calcium influx is often accompanied by parallel increases in the size of active zones and the size of the readily releasable pool of presynaptic vesicles. These changes coincide with homeostatic enhancements of neurotransmitter release. Postsynaptically, there is a great deal of evidence that reduced network activity and loss of calcium influx through CaV1-type calcium channels also results in adaptive homeostatic signaling. Some adaptations drive presynaptic enhancements of vesicle pool size and turnover rate via retrograde signaling, as well as de novo insertion of postsynaptic neurotransmitter receptors. Enhanced calcium influx through CaV1 after network activation or single cell stimulation can elicit the opposite response—homeostatic depression via removal of excitatory receptors. There exist intriguing links between HSP and calcium channelopathies—such as forms of epilepsy, migraine, ataxia, and myasthenia. The episodic nature of some of these disorders suggests alternating periods of stable and unstable function. Uncovering information about how calcium channels are regulated in the context of HSP could be relevant toward understanding these and other disorders.Throughout life, animals face a variety of challenges such as developmental growth, the presence of toxins, or changes in temperature. Neuronal circuits and synapses respond to challenges by executing an array of neuroplasticity paradigms. Some paradigms allow neurons to up- or downregulate activity outputs, while countervailing ones ensure that outputs remain within appropriate physiological ranges. A growing body of evidence suggests that homeostatic synaptic plasticity (HSP) is critical in the latter case. Voltage-gated calcium channels gate forms of HSP. Presynaptically, the aggregate data show that when synapse activity is weakened, homeostatic signaling systems can act to correct impairments, in part by increasing calcium influx through presynaptic Ca Drosophila melanogaster  have emerged as critical for homeostatic control of synapse function in several experimental contexts.Many forms of neuroplasticity drive changes in synaptic outputs, and they are thought to underlie fundamental neurological phenomena, like learning. At the same time, stabilizing forms of neuroplasticity—collectively termed homeostatic synaptic plasticity (HSP)—work to ensure that neuronal outputs are maintained within physiologically appropriate levels. The study of HSP has shed considerable light on how neuronal stability is maintained. Perturbations of synaptic function can trigger homeostatic modulations in activity parameters such as presynaptic neurotransmitter release, neurotransmitter receptor expression, ion channel density, or conductance properties , it is important to consider auxiliary interacting proteins, cellular context, and the high degree to which CaV-driven processes are conserved across metazoans. It is known that neuronal CaV channels mediate cellular calcium entry and regulate activity-dependent processes such as neurotransmission, gene transcription, and intracellular signaling cascades , N- (CaV2.2), and R-type (CaV2.3) calcium channels  is a glutamatergic synapse that exhibits a strong capacity for homeostatic regulation . This phenotype constitutes a block of synaptic homeostasis. One concern about this finding is that any mutant with baseline neurotransmission defects might also have defects in the ability to respond to homeostatic challenges. However, it has been demonstrated that several Drosophila mutations cause baseline NMJ neurotransmission defects without impairing homeostatic plasticity  increases in presynaptic calcium influx, presumably through Cac/CaV2 channels  and S6 kinase (S6K) to potentiate presynaptic release  of presynaptic vesicles  components encoded by the pickpocket11 (ppk11) and pickpocket16 (ppk16) genes are both required for synaptic homeostasis  demonstrated homeostatic changes in presynaptic calcium influx at rodent hippocampal neurons  incorporation of new, GluA2-lacking AMPA receptors postsynaptically; and (2) a retrograde signaling process that results in increased presynaptic release properties, such as spontaneous miniature frequency  regulation during HSP appear to be the cyclin-dependent kinase CDK5 and the α isoform of the phosphatase Calcineurin A (CNAα). Inhibition of CDK5 function in CA1-CA3 hippocampal cultures with the drug roscovitine results in enhanced action potential-evoked release and access to the resting synaptic vesicle pool for release . The kinetics of CaV1 activation can be fast  AMPA receptors to the synapse. To test if CaV1 blockade and AMPA receptor blockade activate a shared mechanism, nifedipine and NBQX were applied concurrently. Dual drug application was indistinguishable from single drug application by biochemical and electrophysiological assays, consistent with a model in which AMPA blockade results in a loss of CaV1 activity  amplitudes also occurs when hippocampal neurons are silenced for 24 h with TTX and allowed to recover in TTX-free saline for a short time before recording , adenylate cyclase 1 activates nuclear transcription of new GluA1 subunits, which are then trafficked and incorporated as homomers —the CaMKK inhibitor STO-609 blocks homeostatic depression  inhibitor I-2. In turn, I-2 phosphorylation is required for appropriate AMPAR trafficking and homeostatic downregulation of synaptic function  antagonists PD98059 and U0126  and neuronal activity-regulated pentraxin  or shrinkage (depression) of dendritic spines in central synapses  and presynaptic CaV2/Cac channels participate in enabling the overgrowth, at the respective stages of bouton budding (DmCa1D) and maturation (Cac)  containing GLR-1 glutamate receptors in the ventral nerve cord (VNC) remains at a set point level, even though absolute synapse number increases dramatically over time  offer a vertebrate model of homeostatic control of synaptic growth gated by VGCCs. In MSNs, increased striatal dopamine causes an increase in MSN spine density  can have debilitating manifestations  and episodic ataxia type 2 (EA2)—result from mutations  in human CACNA1A, which encodes the α1 subunit of presynaptic CaV2.1-type calcium channels  to check if homeostatic plasticity is disrupted.In contrast to FHM1, EA2 is caused by loss-of-function mutations in V2.1-type calcium channels  susceptibility  susceptibility  experience intermittent episodes of muscle weakness and paralysis that last on the order of 3–24 h in length. The attacks often occur upon waking or after high levels of carbohydrate consumption (USNLM/NIH, Understanding how VGCCs help to execute homeostatic forms of neuroplasticity represents a new area of research. It offers a fresh way to consider why VGCCs are regulated in the ways that they are. In healthy neurons, it is logical that calcium channels are co-opted to gate homeostatic signaling processes. Presynaptically, neurotransmitter release is dependent upon calcium influx or local domains of high calcium concentration. Postsynaptically, many signaling processes are dependent upon calcium that enters the cell through receptor complexes and L-type calcium channels. Neurons, synapses, and circuits across metazoan nervous systems are endowed with powerful homeostatic set points of activity, and small adjustments to calcium channel activity parameters could help fine tune synaptic outputs in a variety of ways.V channels interact with intracellular signaling mechanisms and with the activities of other ion channels to control homeostatic set points of excitable cells. What signaling processes directly modulate CaV2 during homeostatic modulations of neurotransmitter release? What postsynaptic cascades are activated or suppressed downstream of calcium influx through CaV1? We have excellent clues at this date from diverse experimental systems reviewed here. Concerning HSP, it will be interesting to see which modes of regulation prove to be universal and which ones prove to be synapse- or organism-specific. With that knowledge in hand, it may be possible to better understand and address numerous phenomena, including CaV channelopathies.An important frontier for research will be to understand how CaThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sceloporus arenicolus) arise in response to variation in landscape pattern within a continuous habitat patch. Our results emphasize links between individual movements at small scales and the emergence of spatial structure within populations which resembles metapopulation dynamics at larger scales. We conclude that population dynamics viewed in a landscape context must consider the explicit distribution and movement of individuals within continuous habitat as well as among habitat patches.Although defining population structure according to discrete habitat patches is convenient for metapopulation theories, taking this approach may overlook structure within populations continuously distributed across landscapes. For example, landscape features within habitat patches direct the movement of organisms and define the density distribution of individuals, which can generate spatial structure and localized dynamics within populations as well as among them. Here, we use the neighborhood concept, which describes population structure relative to the scale of individual movements, to illustrate how localized dynamics within a population of lizards ( Populations of most species are spatially structured at multiple scales Structure may also be found within populations occupying continuous habitats Sceloporus arenicolus), an extreme habitat specialist occurring only in Shinnery Oak (Quercus havardii) sand-dune habitats of southeastern New Mexico and west Texas Here we predict that patterns of population structure thought to manifest regionally in metapopulations also occur at smaller scales within populations due to the response of individuals to landscape pattern within continuous habitats. We explore this prediction using multisite mark-recapture data from a population of the Dunes Sagebrush Lizard (This study was carried out in strict accordance with recommendations in the Guidelines for use of Live Amphibians and Reptiles in Field Research compiled by the American Society of Ichthyologists and Herpetologists (ASIH), The Herpetologists' League (HL), and the Society for the Study of Amphibians and Reptiles (SSAR). Consistent with those recommendations, lizards were marked with toe-clips rather than PIT tags, which require the injection of large objects into relatively small reptiles. Toe-clipping caused only momentary pain and distress, and we rarely encountered any significant bleeding. When bleeding did occur, we applied pressure until the bleeding subsided and then applied triple antibiotic ointment on the wound. This protocol for wild capture and handling of lizards was approved by the Institutional Animal Care and Use Committee of Texas A&M University (Permit Number: AUP 7–159 and 2011–130). All field data were collected on New Mexico public lands. Permits for field work on this state threatened lizard species were approved by New Mexico Department of Game and Fish (Permit Number: 1755).S. arenicolus that varied in landscape pattern .We sampled lizards at Caprock Wildlife Area approximately 48 km east of Roswell, NM. In this area, we selected six sites located within a contiguous patch of Shinnery Oak sand-dune habitat occupied by  pattern . PreviouWe sampled lizards during the peak activity season We characterized landscape pattern across the six sites by mapping their locations on two digitized landcover layers in a GIS. One layer was a classification of vegetation types for sites at a 1-m resolution derived from Landsat Enhanced Thematic Mapper (ETM) satellite imagery and aerial photos In addition to the two-dimensional measures of landscape pattern described above, we quantified three-dimensional measures of blowout heterogeneity that contribute to variation in landscape pattern and have been shown to be important for predicting habitat selection and presence-absence of the lizard at larger scales throughout its range s; probability that a lizard survives from time t to t+1, given it is alive at time t), recapture probability , recruitment rate , rate of population change (λ), and population size (N) . We calculated lizard density by dividing estimates of N by the area  of each grid site. We then used these estimates of lizard density across sites and site-specific measures of lizard movements collected from the subsequent recaptures of individual lizards within grids to calculate neighborhood size (NS) for each site using the equation below:2σ is the variance of intra-grid movements for individual lizards along a single axis  and d is the density of individuals To estimate demographic parameters for each site, we aggregated the data set into monthly trapping occasions, which were then organized into encounter histories. In Program MARK D) is a single metric of population spread that incorporates both the mean and variance in movement distances over time n moves is:il is the length of the i-th move and it is its duration 2/generation; generation time = 1 year; The diffusion rate  demographic rates  and variables describing landscape pattern, 2) demographic rates and neighborhood size, 3) neighborhood size and the variables describing landscape pattern, and 4) demographic rates and diffusion rates across sites.We captured 1,463 lizards over the 5-year mark-recapture study, and 521 of those were recaptures. We used these recaptures to generate 303 encounter histories across all six sites. There were no movements of marked individuals between sites.s), we observed similarities in model structure across the six sites and determined that survival was high for lizards in this region ranging from 0.88 to 0.95 per trapping interval or 0.46 to 0.74 annually (p), we found support for constant model structures at sites 1, 3, 5 and 6 with estimates ranging from 0.21 to 0.31, and for time-dependent structures at sites 2 and 4  and the rate of population change (λ), we observed time-dependent model structures across all sites except site 3, where data supported models with constant structures  as a diffusion threshold for source-sink identity , site 6 as a net importer of individuals or a sink , and site 3 as neutral . Because recruitment estimates do not separate contributions from both birth and immigration rates, we could not directly evaluate whether putative sources or sinks, identified as net exporters or importers of individuals, also exhibited birth rates greater than death rates (b>d), in the case of sources, or birth rates less than death rates (b<d), in the case of sinks. Instead, we used indirect methods to compare birth and death rates across sites based on the following logical arguments. First, by plotting a conservative estimate of lizard population losses  and regional neighborhood connectivity  that generated the source-sink structure we observed are likely best characterized through an explicit understanding of how the spatial configuration of blowouts within Shinnery Oak sand-dune habitats, as well as other factors , directed movements at the level of individual lizards.This conclusion emphasizes the link between individual movements at small scales and the emergence of spatial population structure that can generate metapopulation dynamics at larger scales As our empirical data illustrate how landscape pattern within continuous habitats determines the size of lizard neighborhoods and thus their recruitment rates, it follows that alterations to landscape pattern in these habitats could disrupt neighborhood dynamics. For example, anthropogenic habitat loss and fragmentation could redirect individual lizard movements and cause neighborhoods to be reconfigured with consequences for the structure and persistence of neighborhoods within the population at larger scales. Cell-based simulation models have explored such conservation issues and illustrated how changes in habitat configuration as it relates to individual movements can facilitate or have little effect on population persistence Our study demonstrates that population processes in small-scale neighborhoods reflect those conceived for metapopulations. Our study thus identifies the neighborhood concept as a means to scale individual movements in the landscape up to the dispersal dynamics traditionally reserved for metapopulations. Indeed, this finding illustrates how landscape pattern can generate patterns of structure within populations and is therefore part of a larger synthesis between landscape ecology and metapopulation theory Figure S1Estimates of recruitment and lambda (rate of population change). For the first month on the x-axis “M” = May. Shaded periods on x-axis correspond to trapping occasions. Sites 1–6 are represented by dash-dot, dash-dot-dot, long-dash, dotted, solid, and short-dash lines, respectively. (A) Variable recruitment rates  were observed across sites 1–2 and 4–6, but not site 3. (B) All sites exhibited variation in lambda that was asynchronous across years and sites. No sites exhibited synchrony between recruitment and lambda that would suggest a positive correlation.(TIF)Click here for additional data file.Table S1Locality, year of construction, and number of trapping occasions for 6 sites at Caprock Wildlife Area, NM.(DOC)Click here for additional data file.Table S2Mean (coefficient of variation) of Blowout landcover metrics (meters) for landcover plots surrounding each site calculated in FRAGSTATS. Blowout counts were 45, 45, 21, 29, 47, and 65 for sites 1–6, respectively.(DOC)Click here for additional data file.Table S3Means (coefficient of variation) of three-dimensional and habitat quality metrics measured at each pitfall trap across sites. Blowout counts for the depth metric were 10, 8, 11, 19, 18, and 8 for sites 1–6, respectively.(DOC)Click here for additional data file.Protocol S1Demographic parameter estimation.(DOC)Click here for additional data file.Table S4Model ranking of Cormack–Jolly–Seber (CJS) mark–recapture models estimating apparent survival (s) and recapture probability (p) for S. arenicolus across 6 sites from 2005–09.(DOC)Click here for additional data file.Table S5Model-averaged estimates of apparent survival for female and male S. arenicolus across 6 sites from 2005–09 derived using Cormack-Jolly-Seber (CJS) mark-recapture models.(DOC)Click here for additional data file.Table S6Sex-specific or time-variant (depending on model ranking) estimates of recapture probability for S. arenicolus across 6 sites.(DOC)Click here for additional data file.Table S7Model ranking of Pradel mark–recapture models estimating apparent survival (s), recapture probability (p), and recruitment (f) for S. arenicolus across 6 sites from 2005–09.(DOC)Click here for additional data file.Table S8Model ranking of Pradel mark–recapture models estimating apparent survival (s), recapture probability (p), and rate of population change (λ) for S. arenicolus across 6 sites from 2005–09.(DOC)Click here for additional data file.


Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing.Impaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF- Up to 25% of people with diabetes can expect to develop a foot ulcer at some point in their lives . Due to 2+-independent G-actin binding activity as well as F-actin binding and severing activities [β maturation [One area of research which has been shown to be integral to the wound repair process is that of the actin cytoskeleton which is a filamentous network found in all cells and facilitates processes such as cellular adhesion, migration and contraction , 4. One tivities , 6–8. Intivities . Flii istivities , 11 and tivities , importativities , 14, celtivities , prolifetivities , and infturation . Flii-deturation .β (TRIF), and TRIF-related adaptor molecule (TRAM) [Increased inflammation is an important contributing factor in the failure to heal of diabetic foot ulcers . Inflamme (TRAM) –15. Upone (TRAM)  with woue (TRAM) . κB [κB and increase cytokine secretion [+/−), normal (WT), and high (FliiTg/Tg) Flii gene expression levels to investigate the function of Flii in a murine wound healing model of streptozocin-induced type 1 diabetes. We also investigated whether modulation of Flii by exogenous application of Flii neutralising antibodies improved diabetic wound healing via effects on TLR-mediated inflammation. The LRR region of Flii shares 29% sequence identity and 42% similarity to TLR4 . ThroughκB . Conversecretion . Severalecretion , 16, 17.κB antibody from Abcam . All antibodies were used at a 1 : 100 dilution. The appropriate secondary antibodies were used depending on the fluorescence required—goat anti-mouse Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 were obtained from Life Technologies  and also used at a 1 : 100 dilution. Flii is a highly conserved protein with 95% homology between mice and humans [in vivo diabetic mouse trials were mouse monoclonal anti-Flii antibodies raised against the N-terminus of the LRR domain of the human Flii protein [in vivo diabetic mouse was obtained from Sigma  Mouse monoclonal anti-Flii antibodies raised to the LRR domain of the human Flii protein and the rabbit anti-human MyD88 antibody were obtained from Santa Cruz Biotechnology . Rabbit anti-human TLR4 and TLR9 antibodies were obtained from Imgenex , mouse anti-human CD14 antibody and mouse anti-human CD16 antibody from BD Biosciences , and rabbit anti-human NF-d humans . The Fli protein  and obta+/−), normal (WT), and high (FliiTg/Tg) Flii gene expression levels. Mice lacking one copy of the Flii gene, the double knockout being embryonically lethal [+/−. Transgenic mice carrying two extra copies of human Flii gene incorporated into mouse genome will be written as FliiTg/Tg. All experiments were approved by the Women's and Children's Health Network Animal Ethics Committee following the Australian Code of Practice for the Care and the Use of Animals for Scientific Purposes. Studies were performed using mice with a BALB/c background. Three strains of mice were used in this study with low  was used to induce type 1 diabetes . STZ is toxic to the pancreatic beta-islet cells, rendering the mouse unable to produce adequate amount of insulin. Mice were given one intraperitoneal (IP) injection of STZ for 5 consecutive days (STZ: 50 mg/kg) in citrate buffer of pH 6.5. This dose was chosen based on previously reported studies [Six female Flii studies . Age-matμL of FnAb  or mouse IgG control  immediately after surgery and at 1 and 2 days after wounding. Digital photographs of wounds were taken at days 0 and 7 after wounding. All animals were euthanized at day 7 post-wounding with the wounds harvested, fixed in formalin, and processed for histological analysis. An additional cohort of female WT diabetic mice were injected intradermally around the wound margins with 200 μm) of mouse wounds were cut and stained with haematoxylin and eosin or subjected to immunohistochemistry following antigen retrieval as described previously [κB (1 : 100) were applied and incubated overnight at 4°C. Appropriate Alexa Fluor 488-conjugated secondary antibodies (1 : 100) were used and incubated for 1 hour. Fluorescence intensity was determined using AnalySIS software package , and optical fluorescence in the epidermis and dermis of the wounds was analysed as previously described [Histological sections . Wound size was determined by manually drawing below the clot or the portions of the wound that were not covered by epidermis. Dermal wound gape was determined by measuring between the dermal wound margins. Fluorescent images were taken using an Olympus IX81  at a magnification of ×20. The fluorescent intensity of the staining was calculated using Image Pro-Plus software .t-test or analysis of variance. A P value of 0.05 or less was considered significant.Statistical significance was calculated using a paired Students +/−), normal (Flii+/+), and high (FliiTg/Tg) levels of Flii. Representative digital images of wounds at 7 days subsequent to wounding are shown in +/−; P = 0.01) compared to FliiTg/Tg mice at day 7 following wounding (+/−) compared with Flii overexpressing (FliiTg/Tg) mice (P = 0.05). To assess the biological function of Flii and determine the effect of Flii gene modulation on diabetic wound healing, three lines of mice were used expressing low . Previous studies have shown that intradermal injection of Flii neutralising antibodies reduces the level of Flii in wounds and improves healing . FnAbs w+/−, WT, and FliiTg/Tg mice shows increased Flii in FliiTg/Tg diabetic wounds > WT > Flii+/− wounds . Given that the inflammatory response in diabetic wounds was associated with increased TLR4 expression, we next proceeded to test if the downstream molecule NF-κB production was also affected. κB expression was also increased in Flii overexpressing wounds (5-fold higher than WT diabetic wounds) (P ≤ 0.001FliiTg/Tg versus WT). A reduction in NF-κB was observed between Flii+/− wounds and WT, but this was not statistically significant  whereas NF-κB expression in day 7 FnAb-treated wounds remained unchanged compared to IgG controls (Given that intradermal application of FnAb resulted in improved healing of diabetic wounds , we procin vitro [in vivo [Flii has been identified as a protein that can inhibit the rate of healing by reducing the migration of keratinocytes and fibroblasts and limiting the degree of wound contraction , 22, 28.in vitro  and in v[in vivo . Recent [in vivo . Additio[in vivo , and treκB resulting in cytokine secretion [κB were elevated in the murine diabetic wounds which would further contribute to inflammation and chronicity. In Flii-deficient diabetic mouse wounds, decreased TLR4 and NF-κB were observed and when Flii neutralising antibodies were administered to diabetic wounds, a decrease in TLR4 expression was seen suggesting a dampening of the inflammatory response when Flii levels were reduced. Interestingly, the effect of FnAb was specific to TLR4 and did not appear to affect the production of the proinflammatory cytokine NF-κB suggesting that alternative pathways may still be active in these wounds. Inflammation is an essential component of the normal wound healing process; however excessive inflammation is detrimental to this process . Dispropecretion , and botIn vitro studies have previously shown that the LRR region of Flii shares 29% sequence identity and 42% similarity to TLR4 [in vitro studies showed that through its interaction with MyD88, Flii was able to negatively regulate the TLR4-MyD88-mediated activation of NF-κB and the subsequent cytokine secretion in macrophages [in vivo, in mouse diabetic wounds the increased levels of Flii appear to correlate with an increase in the expression of TLR4 and its signalling protein NF-κB. This is clearly in opposition to the findings in these cell-based studies but may be accounted for by the differing in vivo and in vitro environments. The time points investigated also differ with the in vitro studies looking at responses from 3 to 6 hours whereas this wounding study investigated time points which were measured in days rather than hours. It is, however, not inconceivable that Flii may have a dual role in wounding and in the inflammatory response which depends on the time point investigated.  to TLR4  suggestirophages , 21, ourin vivo studies show that reducing the expression of Flii in diabetic murine wounds improves healing and reduces the proinflammatory response. Being able to manipulate the level of inflammation in a wound would greatly improve the wound healing outcomes of patients with diabetes, and it remains to be elucidated whether neutralisation of Flii in human diabetic wounds could help improve healing of these chronic nonhealing ulcers.In conclusion, inflammation is an integral component of the normal wound healing process and occurs even in the absence of infection; however excessive and prolonged inflammation impairs healing. Flii is a multifunctional protein and is currently emerging as a regulator of inflammation; however, whether it is pro-or anti-inflammatory is still to be determined. Our


Populus tremula × Populus alba) showing that gibberellin (GA) catabolism and repressive signaling mediates shoot growth inhibition and physiological adaptation in response to drought and short-day (SD) induced bud dormancy. Both water deprivation and SDs elicited activation of a suite of poplar GA2ox and DELLA encoding genes. Poplar transgenics with up-regulated GA 2-oxidase (GA2ox) and DELLA domain proteins showed hypersensitive growth inhibition in response to both drought and SDs. In addition, the transgenic plants displayed greater drought resistance as evidenced by increased pigment concentrations (chlorophyll and carotenoid) and reductions in electrolyte leakage (EL). Comparative transcriptome analysis using whole-genome microarray showed that the GA-deficiency and GA-insensitivity, SD-induced dormancy, and drought response in poplar share a common regulon of 684 differentially-expressed genes, which suggest GA metabolism and signaling plays a role in plant physiological adaptations in response to alterations in environmental factors. Our results demonstrate that GA catabolism and repressive signaling represents a major route for control of growth and physiological adaptation in response to immediate or imminent adverse conditions.Survival and productivity of perennial plants in temperate zones are dependent on robust responses to prolonged and seasonal cycles of unfavorable conditions. Here we report whole-genome microarray, expression, physiological, and transgenic evidence in hybrid poplar ( Phenotypic plasticity in response to adverse conditions determines plant productivity and survival. Abiotic stress results in the largest loss in crop yields worldwide Plants reduce growth under adverse conditions as a mechanism to avoid potentially lethal stresses gai and rgl1) with truncation of the DELLA domain are resistant to degradation and impart repressive blocks to several GA-mediated responses Gibberellins (GAs) are involved in regulating several aspects of plant growth and development Arabidopsis thaliana), salt stress leads to DELLA protein stabilization and as a consequence, growth inhibition and increased plant survival XERICO gene, which is involved in drought response An accumulating body of evidence suggests that DELLA domain proteins and GA2oxs are involved in plant abiotic stress response. For example, activation of DELLA domain proteins appears to be crucial for restraining growth in adverse conditions GA 2-oxidase (GA2ox) genes GA2ox genes were shown to be up-regulated GA2ox genes, which in turn leads to reductions in bioactive GAs and suppression of growth GA2ox7 gene In addition to modulation of GA sensitivity, stressful conditions can directly influence levels of bioactive GAs. For example, cold treated Arabidopsis plants have been shown to have increased expression of three Populus, GA-deficient and GA-insensitive transgenics are semidwarfs of varying degrees of severity Populus tremula x Populus alba) constitute a major regulatory circuit mediating growth restraint and physiological adaptation to drought stress and SD photoperiods.Changes in GA catabolism and signaling can have profound effects on tree growth, phenology, morphology, physiological, metabolism, and gene expression PtaGAI1, PtaGAI2, PtaRGL1-1, and PtaRGL1-2) and seven PtaGA2ox (PtaGA2ox1 to 7) encoding genes . Expression of only two (PtaGAI1 and PtaGA2ox2) of the seven genes was specifically influenced by one but not the other treatment  and GA-insensitive (35S::rgl1 and pGAI::gai) transgenic poplar with representative, stable, and intermediate/semidwarf phenotypes  to study their response to drought stress and SD photoperiods. To ensure that any inherent differences in size between genotypes did not obscure analysis of treatment effects, relative growth rates were used to determine significant differences between WT and transgenic plants. Experiments (drought and SD photoperiod) with transgenic plants were performed in a similar fashion to that of expression analysis and as described in the Materials and methods. Prior to implementing drought and SD photoperiod experiments, weekly relative growth rates were not significantly different between transgenic and WT plants under well-watered conditions and long-day photoperiods  in the gai/rgl1 transgenics, as indicated by their significantly decreased growth rates relative to WT in weeks three through five , cease growth after three to five weeks under SD photoperiod We also studied transgenics’ growth response to SD photoperiods that induce winter dormancy. The first response to SDs, which precedes and is a prerequisite for dormancy, is cessation of shoot growth. Poplars are highly photoperiod sensitive, and the genotype under investigation, GA2ox and DELLA expressing transgenic plants differ with respect to their physiological responses to drought, we measured several important parameters before and during drought stress response. We quantified pigment concentrations, photosynthetic rate, transpiration, and stomatal conductance, which are frequently used to measure the degree of stress imposed on plants . The ed to WT . The GA2ed to WT . EL can ed to WT .In support of our quantitative pigment measurements, signs of senescence after water deprivation were more visible in WT leaves compared to transgenics . PercentLeaf senescence preceding winter dormancy is typically initiated by SD photoperiods but is enhanced by low temperatures (<10°C). To investigate senescence in GA-deficient and GA-insensitive plants, we subjected transgenic and WT plants to six weeks of SDs at 21°C followed by three weeks at 4°C and measured the extent of leaf senescence. In a manner similar to their response to drought, all transgenic plants showed delayed senescence when compared to WT .As with bud set, there were no significant differences in the timing of the first bud to flush after winter dormancy between transgenics and WT . HoweverGA2ox overexpressing transgenics, this was followed by a significant increase in lateral branch outgrowth as compared to WT plants . We therefore used whole-genome poplar microarray to compare transcriptomes of transgenic and WT leaves from plants grown under a control environment. We found 2,890 differentially expressed genes  . Gene onLikely because GA is highly integrated into the regulatory network of other hormones GA metabolism effects both cell expansion and proliferation Because our studies suggest that GA-insensitive and GA-deficient plants have faster and more robust responses to dormancy-inducing conditions and drought, we speculated that alterations in GA metabolism and signaling in transgenics may result in transcriptome level changes that are shared with plants that are responding to stress . Therefore, we compared the transcriptome of GA-deficient and GA-insensitive poplars to previously published transcriptomes of WT poplars during dormancy induction and drought response PtaGAI1 and PtaGA2ox2, which were up-regulated by one but not the other factor. Particularly instructive are the trends in expression of the GA2ox genes, for which we have performed a significant amount of previous work GA2ox3 was up-regulated by both drought and SD photoperiods ; furthermore, it was the only gene for which we failed to regenerate RNAi-suppressed transgenic plants, strongly suggesting that this gene is involved in organization of SAM PtaGA2ox3 is likely an important mechanism for controlling growth through modulation of SAM activity. Another gene that was up-regulated by both drought and SDs was PtaGA2ox7. We have previously found that PtaGA2ox7 and its paralog PtaGA2ox2 are predominantly expressed in roots under optimal conditions and positively regulate lateral root proliferation Survival and productivity of long-lived perennial plants in temperate zones are dependent on robust responses to prolonged and seasonal cycles of unfavorable conditions. Here we report whole-genome microarray, expression, physiological, and transgenic evidence suggesting that GA catabolism and repressive signaling mediate shoot growth inhibition in response to both drought and SD-induced bud dormancy. Both water deprivation and SDs elicited up-regulation of a suite of poplar GA2ox and DELLA protein encoding genes  and 2. Woperiods  and 2. PXERICO, which encodes a ubiquitin-ligase that positively regulates ABA biosynthetic encoding genes XERICO was highly up-regulated in the GA-modified transgenic plants. Alternatively the DELLA proteins themselves could be a convergence hub, as an increasing body of evidence suggests that they are focal points of multiple signaling pathways in plants The commonalities in the transcription responses to drought and SDs are interesting because the signaling mechanisms induced by these two types of environmental cues are quite different. One involves a response to osmotic stress Using previously characterized GA-deficient and GA-insensitive transgenics, with increased expression of GA2ox and DELLA protein encoding genes and representative semidwarf phenotypes GA2ox genes is associated with increases in the expression of light-regulated genes In addition to growth cessation, reduction of bioactive GA levels and GA signaling in poplar transgenics significantly modified and/or delayed the onset of several other physiological changes that are typically associated with stress response. For instance, stressful conditions typically cause a reduction in photosynthesis and promote leaf senescence, which in turn negatively affects productivity In addition, carotenoids are antioxidants that can increase stress resistance via detoxification of ROS To better understand the effects of overexpressing GA2ox and DELLA protein encoding genes on drought response, we must also consider the implications of GA-deficient and GA-insensitive transgenics’ semidwarf phenotype. Soil drying experiments can be difficult to interpret because the plants dictate the severity of stress by controlling the balance of water uptake by roots and loss through leaves Because GA-insensitive transgenics responded significantly earlier to water deprivation as compared to WT , it is uGA2ox expressing transgenics have increased branch production which is indicative of decreased apical dominance  and imminent  conditions involving ceasing or reducing growth as well as increasing physiologically acclimative responses to stress. These findings suggest that regulation of GA metabolism and response may be a focal point for evolution of various adaptive strategies in response to short-term and prolonged unfavorable conditions. It also suggests novel venues to engineer and breed for improved stress resistance in crop plants.Populus tremula x P. alba). For consistency, all experiments involving WT plants were performed using this same genotype. Generation of 35S::PcGA2ox, pGAI::gai, and 35S::rgl1 transgenics was previously described Arabidopsis thaliana) DELLA genes gai and rgl1 have complete truncations of the DELLA domains which confers a gain-of-function dominant mutation with constitutive repression of GA signaling. The gai gene was under the control of the native Arabidopsis promoter (pGAI) while rgl1 was under the control of the cauliflower mosaic virus 35S promoter (35S). The GA2ox1 gene was from Phaseolus coccineus (PcGA2ox) and was also under the control of the 35S promoter. Because constructs were previously well-characterized with respect to transgene presence, expression, GA content and stability of phenotype over many years Genetic background for all transgenic plants was the INRA 717-1B clone  and a WT (untransformed control). Plants were randomized and spaced in a rectangular pattern (25×20 cm apart from each other) within pots. The confiding nature of the pots, allowed plants to be grown in a proximity where their roots were exposed to similar conditions to minimize effects of any differences in water usage between genotypes The drought experiment consisted of four genotypes in a completely randomized block design with eight replications. Each block represented a rectangular pot  assigned four plants: one from each of three transgenic genotypes , with three weeks under LDs  followed by six weeks of SDs . Plants were then transferred to a cold room (4°C) for 11 weeks to fulfill the chilling requirement needed for resumption of growth. Finally, plants were transferred to LD conditions and bud flush was monitored on a daily basis.−2 s−1. Instantaneous water use efficiency was calculated as net photosynthesis rate dividing by transpiration rate. Gas exchange measurements were made between 8∶00 and 10∶00 am weekly and at leaf plastochron index (LPI) = 10±1. After five weeks of withholding water, additional measurements were made including percent leaf senescence, percent leaf wilt, and electrolyte leakage (EL). EL was measured with a conductivity meter  using procedures modified from Ren et al. 2 discs obtained from leaves were placed in tubes containing 15 ml of distilled water. Tubes were gently agitated for 4 h and initial conductivity was measured. Samples were then autoclaved for 15 min and conductivity was again measured. EL measurements were repeated three times per plant, and values were averaged and expressed as percentage of initial conductivity. Chlorophyll and carotenoid content was measured on non-stressed (well-watered) plants subjected to drought stress (after withholding water for five weeks). Pigments were extracted in N,N-dimethylformamide according to Porra et al. 2 discs were measured for each plant from LPI = 8±1. Methods similar to those in http://rsbweb.nih.gov/ij/) and the Threshold Colour plugin (http://www.dentistry.bham.ac.uk/landinig/software/software.html).Phenotypic measurements were made on a weekly basis. To avoid problems with soil level changes, all height measurements were made from permanent markers at the base of the plant stem. Net photosynthesis rate, transpiration rate, and stomatal conductance were measured with the portable photosynthesis system LI-6400 , using an air temperature of 25°C and photosynthetically active radiation of 1500 µmol mAll samples used in expression analysis were collected at the same time weekly, immediately frozen in liquid nitrogen, and stored at −80°C for further use. Samples were analyzed in three biological replications, each consisting of tissues pooled from two to three plants. Total RNA was isolated using the RNeasy Plant Mini Kit  with an on-column DNA digestion using RNase-Free DNase (Qiagen). cDNA was synthesized from 2 µg of DNaseI-treated total RNA using Superscript III reverse transcriptase  with an oligo-dT primer. One µl of RT reaction was used for RT-PCR. Primers used in the expression analyses are shown in n+1−Xn)/Xn, where Y is weekly relative growth rate, X is the measured growth parameter, and n is the week.Statistical analysis was performed using SAS 9.1 . In transgenic experiments, treatments consisted of the four genotypes under investigation. For both experiments, analysis of variance (ANOVA) was used to test for overall treatment effects (α = 0.05), and separate ANOVAs were done for each week of the experiment. When significant differences were present, Tukey’s Honestly Significant Difference (HSD) was used to make comparisons among WT and transgenics, or Dunnett’s test (P<0.05) was used when we were interested in making comparison to WT only. For analyses of growth-related parameters, the response variable is the weekly relative growth rate, calculated as: Y =  Transgenic  and wild-type  plants grown in pots (25×20 cm apart from each other) for three weeks under well-watered conditions, prior to being subjected to five weeks under water-withholding conditions. (B) Bars indicate mean±SE of total area (cm3) of at least eight mature leaves per a genotype. (C) Leaf expansion (cm3) was measured weekly under well-watered (week 0) and water-withholding conditions (weeks 1 to 5) on eight ramets/line and eight WT plants. Measurements in C represent the total area of expansion of the first unfurled leaf (leaf plastrochon index 1) after one week. Leaf measurements were made from digital images in ImageJ version 1.43 (http://rsbweb.nih.gov/ij/). Significant differences between transgenic and WT plants were determined by one-way ANOVA followed by Dunnett’s post-hoc test . Scale bar = 25 cm (A).(TIF)Click here for additional data file.Figure S2−3) in pots under well-water (week 0) and water-withholding conditions (weeks 1 to 5).Estimation of volumetric soil moisture content Click here for additional data file.Figure S3Weekly responses of transgenic and WT Populus under well-watered and water- withholding conditions. The dotted line denotes the initiation of water withholding. Red lines show significant differences between weekly responses of transgenics and WT , as determined by one-way ANOVA followed by Dunnett (post-hoc test (P<0.05).(TIF)Click here for additional data file.Table S1Differential expressed genes in GA-modified transgenics compared to WT poplar.(XLSX)Click here for additional data file.Table S2Gene ontology analyses of genes differentially regulated in the GA-modified transgenics.(XLSX)Click here for additional data file.Table S3Gene ontology analyses of genes regulated by the GA modifications, drought and winter dormancy.(XLSX)Click here for additional data file.


FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM. Stem Cells2013;31:1051–1063Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of  Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and is highly aggressive and therapy-resistant –3. Even The transcription factor, FOXM1 is a member of the Forkhead box (FOX) family that consists of more than 50 mammalian proteins with shared homology in the winged helix DNA binding domain. Many genes within this family play critical roles in cell cycle progression and cell fate decision. Initial studies of FoxM1 characterized its role in liver development . IntriguMaternal embryonic leucine-zipper kinase (MELK) is a serine/threonine kinase and is abundantly expressed in GBM and various other cancers. Previously, we found that MELK is highly expressed in GSCs derived from GBM samples and its mRNA expression is inversely correlated with survival in GBM patients . In addiPrior studies have suggested that both FOXM1 and MELK play essential roles in cell cycle progression, cancer cell growth, and maintenance of stem cell state of GBM. Furthermore, MELK and FOXM1 are highly coexpressed and coregulated in GBM suggesting that they are functionally related. Therefore, we sought to test the hypothesis that MELK and FOXM1 directly interact to regulate proliferation of GSC . In thishttp://www.invitrogen.com) supplemented with 2% B27 supplements (Invitrogen) and 20 ng/ml epidermal growth factor (EGF) and fibroblast growth factor-2 , as previously reported  ATP, 2 mM magnesium acetate, and 20 mM dithiothreitol (DTT).FOXM1 cDNA was inserted in enhanced green fluorescent protein (pEGFP)-C1  data were downloaded from the GDS1815 dataset and analyzed for grade III glioma and GBM. The Cancer Genome Atlas (TCGA) data are available through the TCGA Data Portal at http://tcga-data.nci.nih.gov . O. O27]. OFoxM1 mRNA levels were 2.5-fold greater in GBM NS than in normal NS, indicating that FoxM1 expression is elevated in GSCs compared to NPCs in vitro  cells than CD133 (−) cells in both samples  to determine the similarities of the expression profiles between FOXM1 and MELK in high-grade glioma . Significant correlation was also observed in newly diagnosed and recurrent GBM patient samples at RNA levels (Supporting Information p < .0001) with FOXM1 expression. Immunocytochemistry with patient-derived GBM NS (GBM30) exhibited colocalization of FOXM1 with MELK within individual cells  were subjected to EGFP trap, followed by Western blot with MELK or Flag antibody , we examined whether the FOXM1-MELK complex had any effect on mitotic regulators in GBM30 GSCs. Cotransfection of FOXM1 with MELK resulted in a marked elevation of the key mitosis genes .We then investigated the effect of combination treatment with Temozolomide and Siomycin A on GSCs-derived mouse tumors in vivo B–7E. XenFinally, we used the tumor slice culture method to assess Siomycin A treatment on surgical GBM specimens . PatientFOXM1 activity and induces the expression of downstream mitotic regulators. The transcription factor FOXM1 is a master regulator for cell cycle progression and is overexpressed in a number of human cancers including GBM [Molecular signaling between protein kinases and transcription factors plays vital roles in tumor development and maintenance . Here, wding GBM . The proding GBM . Recent ding GBM . FOXM1 iding GBM . Anotherding GBM . This stDespite the advances presented here, open questions still remain. First, it is not clear whether the MELK/FOXM1 protein complex plays a positive role in cancers or cancer stem cells in other organs. Second, it is undetermined whether MELK-mediated signaling is associated with the other proteins that regulate, or are regulated by, FOXM1 in cancer cells. Future studies are needed to elucidate these questions.The cell of origin for GBM is still debatable, although the concept has been evolving. Here, we used a mouse model of gliomagenesis by ablation of key tumor suppressor genes in NPCs causing formation of GBM-like tumors in cerebral cortices. Our data indicated that FOXM1 signaling is operative in NPCs of the developing brains in vivo. Immunohistochemistry exhibited that abundance of FOXM1 protein is progressively elevated as cells progress from the neural progenitor stage through pretumorigenic progenitors to GSC. Sensitivity of GSCs to Siomycin A was markedly higher than that of NPCs. Taken together, these data indicate that GSCs are more dependent on FOXM1 signaling compared to NPCs. However, it remains unknown whether activation of FOXM1 signaling alone is sufficient for gliomagenesis and whether FOXM1 signaling plays a critical role in oligodendrocyte precursor-derived gliomagenesis. Future studies could help address these questions.An important therapeutic implication of the present data is that combined therapy of mouse xenografted tumors with Temozolomide and Siomycin A resulted in diminished NS-forming GSCs in tumors and provided an additional benefit on mouse survival. Although eradication of cancer stem cells appears to be essential for the cure of cancers, recent studies also suggest that non-cancer stem cells acquire cancer stem cell phenotype when challenged by stressors such as ionizing radiation and chemotherapies . To achiIn this study, we demonstrated that the oncoprotein FOXM1 is regulated by the mitotic kinase MELK for its phosphorylation and autoactivation via a direct interaction in GSCs. Although FOXM1 is restrictedly expressed in NPCs in the normal brain and plays a role in NPC growth, a substantial elevation of FOXM1 during gliomagenesis and elevated sensitivity of GSCs over NPCs to pharmacological inhibition of FOXM1 signaling indicate that GSCs are more dependent on FOXM1 signaling for their survival and growth. Thus, targeting the ability of FOXM1 to form a protein complex with MELK may represent a potential therapeutic benefit for GBM. The results presented here may help to gain further insights into the biology of GSCs as well as molecular mechanism of tumorigenesis and therapy resistance in GBM.