Ra) or peak asperity height (Rp) are not well suited to quantifying those counterface damage features in a manner allowing their incorporation into models predictive of polyethylene wear. A diffused lighting technique, which had been previously developed to visualize these microscopic damage features on a global implant level, also allows damaged regions to be automatically segmented. These global-level segmentations in turn provide a basis for performing high-resolution optical profilometry (OP) areal scans, to quantify the microscopic-level damage features. Algorithms are here reported by means of which those imaged damage features can be encoded for input into finite element (FE) wear simulations. A series of retrieved clinically failed implant femoral heads analyzed in this manner exhibited a wide range of numbers and severity of damage features. Illustrative results from corresponding polyethylene wear computations are also presented.Damage to hard bearing surfaces of total joint replacement components typically includes both thin discrete scratches and broader areas of more diffuse scraping. Traditional surface metrology parameters such as average roughness ( Contemporary total hip and total knee arthroplasty  procedures have excellent success rates clinically. However, in a few percent of cases, aseptic loosening due to wear-induced osteolysis remains a major impediment to implant longevity. This is especially a concern for patients with polyethylene bearings whose hard-surface counterfaces have been damaged due to scratching by 3rd bodies , or due Detection of hard-surface damage features has commonly relied upon gross-level visual inspection and optical microscopy . Scanninμm, and they again require long scan times [High resolution maps of surface morphology can be generated through several techniques. Scanning tunneling microscopy transduces surface topography by monitoring the tunneling current flowing between an extremely sharp conductive probe and the sample surface. Atomic force microscopy generates three-dimensional images by means of a probe attached to the tip of a cantilever moving across the surface, monitoring the minute forces of interaction between the sample surface and probe . Both ofan times . More recently, areal measurements of surface morphology have been facilitated by optical profilometry (OP). This technique captures surface features at subnanometer vertical resolution, using light interferometry . (Laser Ra), peak asperity height (Rp), and maximum asperity depth (Rv). While traditional roughness parameters of this class are straightforward to evaluate and interpret, their suitability for quantifying wear-consequential surface damage is less than ideal. For example, Ra is unable to distinguish between large groups of fine scratches versus small numbers of severe scratches. Similarly, Rp fails to differentiate between a single asperity versus multiple asperities of similar height. Also, these traditional roughness measures have normally been reported as isotropic scalar variables. Including the predominant directionality of microscopic-level damage is an important consideration, however, because physical wear tests have shown that differing angles between scratch orientation and the direction of relative surface motion can produce order-of-magnitude differences in wear rate elevation [The measurements that are output from profilometry scans most commonly have been standard surface roughness parameters such as average roughness (levation . Moreovelevation .Rp (peak asperity height). Even empirically, however, it has been difficult to identify statistically significant wear-versus-roughness relationships for the complex articulations characteristic of in situ function of whole implants. For example, in a study of 35 retrieval implants [r = 0.374, P = 0.099) between clinical wear factors and Ra, with corresponding Rp values having an even weaker relationship with wear . While the microtopography of the hard-surface counterface is clearly a major influence on the rate of polyethylene wear , direct implants , there wIn order to move beyond empirical observations, and to help underpin physics-based models of damage versus wear, it is essential to quantify the severity and directionality of individual scratches and scrapes. Novel image-based computational techniques developed for that purpose are here reported. These computational techniques for surface damage registry are applied to a series of total hip femoral heads that had been surgically retrieved following implant clinical failure. Characteristic aspects of whole-surface damage severity are reported for these retrievals. Finally, results are presented from an illustrative finite element computation of polyethylene wear acceleration associated with the damage to a specific femoral head from this series. 2) were then taken from the polar direction and at 30 degree increments circumferentially, so as to image the entire bearing surface. These images provided vivid visual rendition of all macroscopically apparent damage features on the entire implant bearing surface.  The associated  intensity modulations provided a basis for the damage to be objectively registered for purposes of image analysis. The damage was of two principal types: scratches and scrapes. The distinction was that scratches were manifest as thin discrete individual darkened lines, whereas scrapes were manifest as broad swaths of diffuse darkening, within which individual scratch tracks could not be distinguished at the global image level. Retrieval femoral heads were first digitally photographed using a novel diffused-light illumination technique . This inen masse data processing in this retrieval series. A custom-written MATLAB routine was used to determine regions of damage apparent in the global-level images. Canny edge detection was first performed to detect regions of damage, based on grayscale discontinuity relative to (bright) undamaged regions. This edge detector distinguished edges on the basis of maximum gradients of intensity. Edges were flagged if their gradients fell above an analyst-set threshold. This threshold value was set such that it was sensitive enough to detect the fine-scale scratches, but high enough so as to avoid influence from grayscale variations associated with the necessarily nonuniform distance from the camera lens. Next, a median filter was applied to the original images, replacing each pixel's intensity with the median of pixel intensities in a surrounding square region of analyst-specified size. Median filtering in this context had the effect of “blurring away” linear damage features  whose breadths were below a specific threshold. Analyst specification of the median filter size thus provided a basis for objectively distinguishing between scratches versus scrapes, for which different computational treatments were utilized for purposes of polyethylene wear modeling (see below). Empirically, various filter sizes were applied to representative original images that contained both scratches and scrapes . A 20 × The darkened regions remaining after the median filtering operation were then autosegmented using an analyst-set intensity threshold. These constituted the scrape features. The scrape regions thus identified were then removed from the pre-median-filtered Canny edge detection result, leaving the remaining  damage features to be classified as scratches . A Houghμm2 per pixel, and the maximum out-of-plane resolution was 0.01 μm. The instrument was capable of directly imaging individual areas of sizes up to ~1 mm × 1 mm, and it could autostitch those captured individual images into composite images sized up to ~5 mm × 5 mm. The instrument's internal software allowed for removal of the global  curvature that was present in the raw scans. The parameters of global spherical curvature were estimated using a least-squares-error algorithm to achieve a quadratic fit to the raw surface data. Removal of this spherical form from the raw data therefore enabled determination of local deviations from the native implant surface. The internal software also included capability to report standard surface roughness parameters . Next, OP was used to quantify the severity of each scratch or scrape. A Veeco Contour GT noncontact profiler , which captured surface features by means of light interferometry, was employed for this purpose. The maximum resolution in a plane tangential to the target surface was 0.25 Superimposition of analyst-selected control points allowed alignment of the OP scans with their respective global-level images Figures . For eacEncoding the identified damage features involved accounting for both directionality and severity. In the case of scratches, directionality was simply the locally prevailing orientation of the scratch, and severity was based on the average scratch lip height (the dominant factor in wear elevation ) calculaRa) value within each scrape polygon was simply the average of the height differences of individual pixels, relative to the mean surface height of all the pixels within the scrape polygon. In the case of scrapes, the directionality of interest for purposes of polyethylene wear modeling was not the macro-level direction of the scrape, but rather the direction of “microlevel” scratching within the scrape. While these two orientations were similar or nearly so for many scrapes, there were notable exceptions, such as when circumferentially oriented macroscrapes resulted from transverse sliding during egress of edge-loaded femoral heads during subluxation. To identify the direction of microscratching, a Hough transform was performed on each scrape OP dataset, which detected the microscratch lines within the scrape. The longest such line was designated as the scraping direction for that scrape area . SeverithL) and the scaling factor (kinc) for wear coefficient elevation, the specific parameters beingμm. For the case of scrapes, the Ra values were converted to wear coefficient scaling factors using a power law relationship [Ra measurements in μm.Current computational models of polyethylene wear in total joint replacements most commonly implement some form of the classic Archard wear formula . This fotionship , the speRa values) are reported in Tables μm. In the interest of completeness, the present set of results includes scratches with lip heights as low as 0.07 μm. This may be unnecessarily exhaustive, however, since there is experimental evidence that the lip height threshold for detectable wear rate acceleration from individual scratches is substantially higher, on the order of 1 μm [Scratch and scrape damage was encoded for nine representative specimens from our institution's collection of femoral head retrievals . The ima of 1 μm . If desiRa values of scraped regions also showed considerable variability (μm (specimen no. 8), a three-order-of-magnitude elevation relative to typical Ra values for undamaged implant surfaces [ The computed iability . Series-surfaces .These damage features can produce substantial increases in local wear rates. The presence of the most severe scratches increased the local wear coefficient by a factor of approximately 50, as shown by . The mosRa values, along with their associated individual directionalities, demonstrates a range of damage features on typical retrieval femoral heads. While the present OP data captures were similar to those for other OP applications [This collection of scratch hip heights and scrape ications , 21, theications . Illustratively, an FE wear model of one particular specimen (specimen no. 6) from this series was generated by mapping each identified damage feature's severity, orientation, and global-level geometry onto the femoral head. Collectively, these damage features, comprised of a total of 211 scratch segments and 68 scrape regions, led to a 3.8-fold increase in polyethylene liner wear, compared to a baseline simulation of an otherwise-identical undamaged femoral head . Of thisWear rate increases computed using this technique have been physically validated both for isotropically roughened patches  and for μm, and severe damage features with scratch lips approaching 4 μm. The present series was restricted to total hip femoral component retrievals. This same damage registration framework presumably could also be used to characterize hard counterfaces in other total joint replacements such as the femoral components of total knees, which often exhibit damage features similar to those catalogued here. For example, a 2D interferometry study of TKA retrievals  identifiQuantification of femoral head damage through this analysis framework relies on the global diffused-light digital photographs and corresponding edge detection computations to identify damage features. While the diffused lighting technique in almost all instances provided much more vivid rendition of damage features than was apparent visually under room lighting, there were several instances where fine scratches that were visually apparent under normal room lighting were not visible in the diffused-light images. The OP scans of such “disappearing” scratches showed that these fine scratches had extremely low lips, indicative of their not being critical in terms of the end goal of determining wear rate acceleration. Further work to elucidate the relationship between diffused-light image grayscale values versus corresponding scratch lip heights may offer insight in this seemingly anomalous phenomenon.For total joint replacements with a polyethylene bearing surface, dramatically accelerated wear is often associated with accrual of scratch or scrape damage to the hard-surface counterface. Moving from qualitative to quantitative assessments of this interaction requires a basis for representing hard-surface damage in a manner conducive to performance of physics-based wear analyses. The present paper reports a multiscale experimental/computational framework for making such damage representations. Global-level and microlevel imaging are coupled to computationally register the severity and directionality of both scratch and scrape damage present on entire implant bearing surfaces. This damage registry framework proved practical for use for typical retrieval total hip implants, thus opening the way for quantitative analyses of damage-related polyethylene wear rate acceleration on a case-specific basis.To the authors' knowledge, the present datasets constitute the first-ever compilations of whole-surface damage features on orthopaedic total joint replacements. Besides their usage in the context of enabling wear computations, such datasets will likely prove useful in other contexts, such as for forensic assessment of specific surface damage events, and “reverse engineering” of implant designs to minimize wear-consequential counterface damage.


To test if CYLD is involved in ciliogenesis, we studied the presence of motile cilia in the trachea. Cyld (del17/del17) embryo tracheas exhibit a decreased number of ciliated cells compared to wild type ones of the same litter. In addition, these ciliated cells have fewer and shorter cilia. Similar results were obtained on ependymal cell culture. Transmission electron micrographs demonstrate that most of the basal bodies fail to anchor to the plasma membrane. We demonstrate that in MEFs derived from cyld (del17/del17) embryo, CYLD and CAP350 do not longer interact in contrast to MEFs derived from wild type embryo. In addition, these mutant MEFs exhibit primary cilia growth defect compared to MEFs derived from wild type mice.The tumor suppressor CYLD gene encodes a deubiquitinating enzyme that removes lys-63-linked ubiquitin chains. CYLD regulates, by its catalytic domain, NF-kB, c-Jun kinase and Wnt/β-catenin signalling pathways. When mutated in its catalytic domain CYLD cause skin appendages tumors as in familial cylindromatosis. We have found CYLD as a partner of the centrosomal protein CAP350 in immunoprecipitation experiments followed by mass spectrometry. CAP350 was shown to play a role in microtubule stabilization . Here, we show that CYLD localizes to the centrosome in cells and is enriched in purified centrosomes. To understand the functional interaction between CYLD and CAP350, we studied mice carrying deletion of part of the deubiquitinase domain and mimicking the human pathology. These homozygous mice


Recovery from aneurysmal subarachnoid hemorrhage (SAH) is often incomplete and accompanied by subtle but persistent cognitive deficits. Previous neuropsychological reports indicate these deficits include most prominently memory impairment, with working memory particularly affected. The neural basis of these memory deficits remains unknown and unexplored by functional magnetic resonance imaging (fMRI). In the present study, patients who experienced (SAH) underwent fMRI during the performance of a verbal working memory paradigm. Behavioral results indicated a subtle but statistically significant impairment relative to healthy subjects in working memory performance accuracy, which was accompanied by relatively increased blood-oxygen level dependent signal in widespread left and right hemisphere cortical areas during periods of encoding, maintenance, and retrieval. Activity increases remained after factoring out inter-individual differences in age and task performance, and included most notably left hemisphere regions associated with phonological loop processing, bilateral sensorimotor regions, and right hemisphere dorsolateral prefrontal cortex. We conclude that deficits in verbal working memory following recovery from (SAH) are accompanied by widespread differences in hemodynamic correlates of neural activity. These differences are discussed with respect to the immediate and delayed focal and global brain damage that can occur following (SAH), and the possibility that this damage induces subcortical disconnection and subsequent decreased efficiency in neural processing. Subarachnoid hemorrhage (SAH) due to aneurysm rupture accounts for about five percent of all strokes and affects approximately 30,000 individuals annually in the United States –3. In coTraditionally, aneurysmal SAH has been associated with high mortality and morbidity, but the last few decades have seen a tremendous improvement in overall care of such patients. Associated mortality has fallen by about 50% in the last two decades with case fatality rates improving at 0.9% every year. Interestingly, this has not been associated with a rebound increase in the proportion of survivors with severe neurological disabilities . Still, Although most published outcome data from survivors of aneurysmal SAH place patients with mild disabilities in a “good recovery” category , severalThe physical, economic, and emotional toll of SAH is so dramatic –16 becauThere are several questions that remain to be answered about impaired memory function in SAH patients. From a behavioral perspective, it remains to be determined what aspects of short-term memory, particularly working memory , are mosIn the present study, we undertook an exploratory behavioral and neuroimaging investigation of SAH patients with two main objectives. First, we examined verbal working memory using a well-validated task paradigm in order to measure the accuracy and speed of working memory processing. Second, using this task we examined using 3 T functional magnetic resonance imaging (fMRI) the blood oxygenation level dependent (BOLD) activity during the three temporal phases of working memory encoding, maintenance, and retrieval. Differences between patients and controls in the fMRI-BOLD activity patterns and their covariation with measures of age and performance are presented, followed by discussion and some preliminary conclusions regarding the behavioral and neural correlates of verbal memory impairment in SAH.Eleven patients who experienced SAH participated in this study. Detailed demographic and clinical characteristics for each patient are summarized in Table Each participant completed eight blocks of a variant of a Sternberg  verbal wAn 18 s control period was placed between the sequence of eight working memory trial blocks to allow for fMRI signal to return to baseline before a new encoding period started. To control for visuomotor processing unrelated to working memory processing, the baseline period consisted of change detection in the background color of sequentially presented crosses. A total of seven crosses appeared during each baseline period with six appearing on a white background and with one appearing randomly on a green background. Participants were instructed to press a button when a cross on the green background appeared during the center fixation.Brain scans were obtained with a 3 T Philips Intera scanner  equipped with an eight-channel head coil (SENSE acquisition). A high-resolution 3D T1-weighted magnetization prepared rapid acquisition turbo field sequence was acquired . During working memory task performance, functional images were obtained with a gradient-recalled echo-planar imaging sequence sensitive to blood-oxygen level dependent contrast .All image analysis was performed using tools in the freely available AFNI software package , 33. Fort-statistic maps were computed and thresholded with recommended  for each group of controls and patients . MEMA t-maps were also computed for the difference between patients and controls for each processing period . Finally, t-maps were computed summarizing at each voxel how the BOLD-fMRI signal fluctuated differently as a function of age and performance accuracy between the patients and controls. The within- and between-group activity and covariate maps were projected in different colors as a function of processing period to pial and white/gray matter border surface reconstructions of the TT_N27 brain in MNI space for visualization of whole-brain differences.For each working memory period  relative to the control baseline, voxelwise ommended  parametep < 0.003, Figure p = 0.42).Patients performed significantly worse  BOLD-fMRI activity across all working memory processing periods  compared to younger adults at similar levels of difficulty. An idea to explain this is the Compensation-Related Utilization of Neural Circuits Hypothesis  whereby To conclude, the present study provides the first fMRI evidence of working memory impairment following recovery from SAH. The behavioral finding of subtle but significant impairment in task working memory task performance is accompanied by increased BOLD-fMRI activity across widespread brain areas and during the encoding, maintenance, and retrieval periods. Future investigations should compare larger samples of SAH patients to age-matched patients who have mild cognitive impairment, those at risk for Alzheimer’s disease, and those diagnosed with Alzheimer’s disease in order to identify with more specificity how the pattern of brain damage unique to SAH affects working memory, and to relate the altered functional activity profiles to underlying structural connectivity changes.Designed the study: Timothy M. Ellmore; Collected the data: Timothy M. Ellmore, Fiona Rohlffs, Faraz Khursheed; Analyzed the data: Timothy M. Ellmore, Fiona Rohlffs, Faraz Khursheed; Wrote the paper: Timothy M. Ellmore, Faraz Khursheed.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Preclinical data on putative CSC targets are emerging by now. These preliminary studies are critical for the next generation of lung cancer therapies.Lung cancer remains the leading cause of cancer death. Understanding lung tumors physiopathology should provide opportunity to prevent tumor development or/and improve their therapeutic management. Cancer stem cell (CSC) theory refers to a subpopulation of cancer cells, also named tumor-initiating cells, that can drive cancer development. Cells presenting these characteristics have been identified and isolated from lung cancer. Exploring cell markers and signaling pathways specific to lung CSCs may lead to progress in therapy and improve the prognosis of patients with lung cancer. Continuous efforts in developing  Despite important improvements in therapies over the last decades, lung cancer prognosis remains very poor. After apparent successful initial therapy, development of secondary tumors often leads to a lethal relapse. Substantial growing experimental evidence has suggested that many cancers, including lung cancer, may be driven by a small subpopulation of self-renewing cells that could sustain malignant growth . InnovatTwo major models have been described for tumor propagation: the clonal evolution model, involving a stochastic component, and the CSC model defined as hierarchical. According to the clonal evolution model, neoplasm arise from a single cell of origin, and tumor progression results from acquired genetic variability within the original clone allowing sequential selection of more aggressive sublines . CSC mod2 gradients . Th. Th74]. rognosis , 103. Iner cells . Sox2 is a critical transcription regulator of embryonic stem cells [Sox2 controls self-renewal and differentiation processes [Sox2 has been proposed to be an oncogene which expression is essential to lung SCC carcinogenesis and which is capable of transforming and conferring tumor initiating properties to human lung squamous cells [Sox2 overexpression seem to have a worse prognosis [em cells . Sox2 corocesses . It is arocesses , 58. Soxus cells . Additiorognosis .Oct4 and Sox2 are two of the four crucial transcription factors capable of cooperating to reprogram differentiated cells into an induced pluripotent stem cell-like phenotype [Oct4, Sox2, c-Myc, and Klf4 can reprogram a somatic genome back into an embryonic stem cell status. Is there a comparable way for cancer cells to reacquire an immature profile with properties of self-renewal? Oct4 and Sox2 may participate to a similar process leading to lung CSCs.henotype , 110. Nuc-kit has been shown to operate as a SCF receptor promoting tumor growth [c-kit have lower survival rates and show resistance to chemotherapy [c-kit signaling pathway inhibits CSC proliferation and survival after chemotherapy exposure in human lung cancer cell lines [c-kit axis blockade by SCF-neutralizing antibodies or by imatinib (Gleevec), an inhibitor of c-kit, combined to cisplatin therapy might lead to inhibition of both non-CSCs and CSCs.Stem Cell Factor (SCF) is a mitogenic and angiogenic factor involved in carcinogenesis. Human r growth –113. Patotherapy . Blockinll lines . ExposurDifferences between normal stem cells and CSCs may provide novel antigenic and molecular targets for therapy . It is iRunx3, Oct4, Sox2, and c-kit in lung tumor differentiation and treatment resistance. Further in vitro and in vivo studies are needed to design combined therapies in order to determine whether these candidates are potential targets for new therapeutic strategies against lung cancer. Moreover, the prognostic and predictive value of CSCs identification from tumor biopsies or among circulating tumor cells remains to be determined.CSC is central to cancer cell biology and cancer therapy. The knowledge of CSC signaling pathways may lead to new therapies able to eradicate lung CSC or induce differentiation of these cells. Promising models have recently been developed to isolate the subpopulation of CSCs within the lung-differentiated cells of the tumor mass. These models are useful tools to better characterize lung CSCs. Targeting the CSC or its microenvironmental niche could inhibit self-renewing and overcome chemo-radio resistance. Some consistent preclinical data are emerging on the role of transcriptional factors such as


Dynamic cerebral autoregulation (dCA) is impaired following stroke. However, the relationship between dCA, brain atrophy, and functional outcomes following stroke remains unclear. In this study, we aimed to determine whether impairment of dCA is associated with atrophy in specific regions or globally, thereby affecting daily functions in stroke patients.We performed a retrospective analysis of 33 subjects with chronic infarctions in the middle cerebral artery territory, and 109 age-matched non-stroke subjects. dCA was assessed via the phase relationship between arterial blood pressure and cerebral blood flow velocity. Brain tissue volumes were quantified from MRI. Functional status was assessed by gait speed, instrumental activities of daily living (IADL), modified Rankin Scale, and NIH Stroke Score.Compared to the non-stroke group, stroke subjects showed degraded dCA bilaterally, and showed gray matter atrophy in the frontal, parietal and temporal lobes ipsilateral to infarct. In stroke subjects, better dCA was associated with less temporal lobe gray matter atrophy on the infracted side ( Cerebral autoregulation (CA) modulates cerebral blood flow in order to meet regional perfusion demands despite variations in arterial blood pressure (BP) associated with daily activities Both impaired dCA Noninvasive assessment of dCA often entails examining the coupling between continuous BP and cerebral blood flow velocity (BFV), measured by transcranial Doppler ultrasound (TCD). However, finding computational methods for the accurate quantification of this relationship is a challenge to reliable dCA assessment. Multimodal pressure-flow (MMPF) analysis This study applied the MMPF-derived dCA measure to examine the relationship between dCA, regional brain tissue volumes, and functional status in a retrospective analysis of elderly subjects with chronic large vessel infarctions in the middle cerebral artery (MCA) territory, and in age-matched non-stroke subjects. We hypothesize that worse perfusion regulation is associated with enhanced gray matter atrophy in the temporal lobe, and worse long-term functional status in the elderly with chronic ischemic infarctions.All subjects signed informed consent and the study was approved by the Institutional Review Board at Beth Israel Deaconess Medical Center (BIDMC). Participants were recruited from community advertisement, Beth Israel Deaconess Medical Center, Joslin Diabetes Clinic patient registries and from the Harvard Cooperative Program on Aging research subject registry.The data for this retrospective analysis of 142 subjects were selected from a database of records prospectively collected at the Syncope and Falls in the Elderly Laboratory and the Magnetic Resonance Imaging Center at BIDMC. The database was composed of records from three completed projects spanning January 2002 to February 2008: Cerebral vasoregulation in the elderly with stroke (March 2003–April 2005); Cerebral vasoregulation in diabetes (January 2002–December 2005); and Cerebral perfusion and cognitive decline in type 2 diabetes (January 2006–December 2008). Grant numbers and awarding institutions are provided in the financial disclosures section. All stroke subjects included in the current project were recruited for the vasoregulation in the elderly study while diabetic non-stroke subjects were from the vasoregulation in diabetes and cognitive decline in diabetes studies. Non-diabetic non-stroke subjects were recruited in all three studies. The subjects were selected for the present cohort, only if they completed both TCD and MRI measurements, and met the inclusion/exclusion criteria detailed below and in Included stroke subjects had large vessel hemispheric MCA infarcts affecting All diabetic subjects were required to be diagnosed with type-II diabetes mellitus (DM) and to have been treated for at least 1 year prior to participation. Non-diabetic controls were age- and sex- matched to diabetic and stroke subjects from their respective studies with no clinical history of stroke and no focal deficits on neurological examination. Non-diabetic participants were required to have normal fasting glucose.Subjects were excluded if they had intracranial or subarachnoid hemorrhage on MRI or CT or carotid artery stenosis . Other exclusion criteria included myocardial infarction within 6 months and other clinically important cardiac diseases; arrhythmias; significant nephropathy; kidney or liver transplant; renal or congestive heart failure; type I DM; or neurological or other systemic disorders. Incompatibility with 3 Tesla MRI, including claustrophobia, metal implants, pacemakers, and arterial stents was also an excluding factor.Collectively, the three studies recruited 358 subjects . We excluded 145 subjects  because they either withdrew consent, they met exclusion criteria (listed above), or they did not get permission from their primary care provider. Of those subjects excluded, 24 subjects  were excluded due to a poor temporal insonation window. From the remaining 213 subjects, 142 of them  had complete TCD and MRI recordings and were used for the final analysis in the present study. Subjects with diabetes, hypertension, or both were included as part of the non-stroke group in order to control for dCA impairment associated with risk factors related to stroke. Demographic information of the selected cohort is listed in The stroke group consisted of 33 subjects that were 6.712 (CO2R) was measured using 3 minutes of hyperventilation followed by 3 minutes re-breathing 5% CO2 in an air bag. Vasoreactivity was calculated as the slope of the regression of CO2 on BFV over baseline, hyperventilation, and rebreathing conditions. BFVs in both MCAs were measured from trans-temporal windows using TCD . BP was recorded from the finger using the volume-clamp technique with a Finapres device  and corroborated by sphygmomanometer measurements. BP, BFV, respiration and end-tidal CO2 measurements  were recorded at 500 HZ. Signals were decimated to 50 Hz before analysis.Experiments were conducted in the morning after a thirty-minute rest during instrumentation. Baseline recordings of 5–10 minutes were collected during resting conditions when subjects were supine, awake and breathing regularly at their normal respiratory frequency. Vasoreactivity to CO3/cm3) of GM, and WM were used for analysis. Vessel diameters were derived from 3D MR angiography  using the Medical Image Processing, Analysis, and Visualization (MIPAV) software from the Biomedical Imaging Research Services Section, NIH, Bethesda, MD, at 3 locations and averaged. Diameters Internal carotid arteries (ICA) and MCAs were computed from a single-slice transverse view with conservatively estimated accuracy . Anatomical 3D magnetization prepared rapid gradient echo (MP-RAGE) images were used to quantify brain volumes with the Statistical Parametric Mapping software  using spatial normalization and tissue classification. An anatomical template  was applied to measure GM and white matter (WM) in frontal, temporal, parietal, and occipital lobes. Normalized volumes , Instrumental Activities of Daily Living Multimodal pressure-flow (MMPF) analysis was used for non-invasive assessment of dCA. Details on the development and performance of the method have been published previously The MMPF analysis for this study was performed according to the following four major steps:Decomposition of BP and BFV signals into multiple empirical modes.Central to the MMPF method is the Hilbert-Huang transform Selection of empirical modes for dominant oscillations in BP at 0.1–0.3 Hz and corresponding oscillations in BFV.In order to determine a meaningful phase relationship between the BP and BFV time series, empirical modes from both series must be selected from within the same frequency band. Previous studies have shown that dCA can be assessed from respiratory-induced pressure-flow variations during spontaneous respiration Calculation of instantaneous phases of extracted BP and BFV oscillations.Since each empirical mode has zero mean and is sufficiently narrow-band, the complex part of each mode can be calculated by the Hilbert transform Calculation of the mean BP-BFV phase difference  and a Ònon-strokeÓ side (RND2). The side assignment was implemented in order to have a left-side-stroke/right-side-stroke probability that approximately matched the distribution of the stroke group. Univariate group differences were determined by one -, or two-tailed t-test, or To constrain the number of variables under consideration, regression analyses were limited to those areas directly affected by stroke (i.e. MCA territory on the stroke side/RND1). Linear regression models were tested for the effects of Regression parameters were estimated using the traditional least squares estimator as well as the Theil-Sen robust regression estimator www-rcf.usc.edu/rwilcox/ on 6/19/11) for the statistical computing software R Robust regression parameter inference, including simultaneous 95% confidence intervals and hypothesis test statistics, was estimated by bootstrapping of observations. For each robust regression, bootstrapping consisted of n = 600 resamples of the stroke group, with replacement, of the multivariate observations. Regression parameter estimates, standard errors, bootstrap confidence intervals, and hypothesis test statistics were calculated using functions written by R.R. Wilcox Relationships between GM and IADL and between For groups of hypothesis tests that included multiple variables (differences between groups and between sides), the threshold of significance was adjusted to maintain a false discovery rate 2 vasoreactivity and laboratory results were similar between the stroke and non-stroke groups   after coIn contrast with our findings of associations between Similar to our results with least squares regression, the Theil-Sen estimate for a relationship between gait speed and temporal GM was marginally significant, (IADL was not significantly associated with temporal GM (Lack of fit: In order to examine the confounding effect of infarct volume on linear regressions observed above, linear regressions of infarct volume on gait speed and temporal lobe GM were examined, after correcting for age, sex, mean BP and BMI. Least squares regression showed that larger infarct volumes were associated with significantly slower gait speed (model fit: This study examined the relationships among dCA, brain structural volumes, and functional status in subjects with chronic ischemic stroke using a nonlinear dCA assessment computed using the MMPF method. Both traditional least squares regression and robust regression were used to test the hypothesis that better dCA function is associated with less GM atrophy and better functional status. Supporting this hypothesis, smaller Poor clinical outcomes have been shown to be associated with impaired dCA following both brain injury Atrophy of brain tissue continues following the acute stroke period and extends from periinfarct zones to contralateral and remote cortical and subcortical regions that are functionally connected to the infarct site Both impaired CA In the present study, although all MCA-territory brain regions showed significant atrophy for stroke subjects, the relationships between dCA impairment, GM atrophy, and functional status were most prominent for temporal lobe GM. These relationships were not observed in age-matched, diabetic, or non-diabetic non-stroke subjects, and were independent of BP. However, diabetic subjects also had impaired dCA compared to healthy nonstroke subjects, which suggests that associations between dCA, functional impairment, and temporal lobe GM are stroke-specific and not due to normal aging or stroke-independent dCA impairment.Temporal lobe structures such as the insular cortex and amygdala are key centers of the autonomic network Disrupted autonomic regulation after stroke may also influence the clinical outcomes measured in this study, independent from dCA. Sympathetic reflex activity is attenuated with chronic stroke and this attenuation is correlated with functional motor capacity We hypothesize that treatments aimed at the improvement of dCA may play a role in optimizing the functional performance and quality of life in elderly people with chronic ischemic stroke. Therefore, the dCA status and the potential effects of treatments (such as antihypertensive medications The results of least squares regression were in agreement with robust regression estimates for the relationship between Including infarct volume in the linear models had a considerable effect on robust regression results. When including infarct volume, the regressions of 2 reactivity between groups were detected (2 reactivity for stroke subjects 2 reactivity was lower in stroke subjects than in non-stroke subjects. However, the between-group difference was not significant due to the large standard errors within both groups.The size and variability of the sample may also explain why no significant differences in COdetected . In agreDue to the retrospective nature of the present study, the sample selected for analysis was not population-based. Specifically, our non-stroke group included individuals with diabetes, hypertension, or both while all stroke subjects were non-diabetic. However, diabetes is a major risk factor for stroke and hypertension The study design has also limited the elucidation of causality between impaired cerebral autoregulation, brain tissue loss and poor functional outcomes in stroke patients. Since stroke itself can cause impairment of dCA, brain tissue loss and worse functional outcomes, the cross-sectional study design cannot demonstrate a causal link between dCA, tissue loss and functional outcome. Therefore, future work should include a longitudinal study that would evaluate the time course and relationship between cerebral autoregulation and functional outcomes.There are likely associations between dCA, temporal lobe GM, gait speed, and IADL, indicating that dCA may impact GM atrophy and functional recovery following stroke. The relationships between dCA, temporal lobe GM and functional status were independent of age, sex, BMI, mean BP and mean BFV, but it is unclear how infarct volume is associated with these parameters. Therefore, dCA impairment may be an important factor underlying perfusion adaptation to daily activities and progression of regional atrophy and functional recovery in patients with stroke.


Sleeping Beauty DNA transposon as a powerful engine, a growing list of transposable elements with activity in human cells have moved into biomedical experimentation and preclinical therapy as versatile vehicles for delivery and genomic insertion of transgenes. In this review, we aim to link the mechanisms that drive transposon evolution with the realities and potential challenges we are facing when adapting DNA transposons for gene transfer. We argue that DNA transposon-derived vectors may carry inherent, and potentially limiting, traits of their mother elements. By understanding in detail the evolutionary journey of transposons, from host colonization to element multiplication and inactivation, we may better exploit the potential of distinct transposable elements. Hence, parallel efforts to investigate and develop distinct, but potent, transposon-based vector systems will benefit the broad applications of gene transfer. Insight and clever optimization have shaped new DNA transposon vectors, which recently debuted in the first DNA transposon-based clinical trial. Learning from an evolutionary drive may help us create gene vehicles that are safer, more efficient, and less prone for suppression and inactivation.DNA transposons are primitive genetic elements which have colonized living organisms from plants to bacteria and mammals. Through evolution such parasitic elements have shaped their host genomes by replicating and relocating between chromosomal loci in processes catalyzed by the transposase proteins encoded by the elements themselves. DNA transposable elements are constantly adapting to life in the genome, and self-suppressive regulation as well as defensive host mechanisms may assist in buffering ‘cut-and-paste’ DNA mobilization until accumulating mutations will eventually restrict events of transposition. With the reconstructed  Since its birth the field of therapeutic gene transfer has travelled on a rough road from initial optimism through hype and disappointment towards scientifically well-founded hope and signs of clinical applicability and success. Indeed, incipient confidence is fuelled by promising preclinical and clinical findings and by an increasing wealth of improved gene transfer technologies. Continuous focus on gene carriers and their biological properties, immune responses, and interactions with host cells already has provided clinical benefit -3 and wiSleeping Beauty DNA transposon in the driver’s seat, transposable DNA elements have emerged as promising nonviral vehicles for persistent gene delivery P elementson ends . After eson ends ,69, and ootprint . Also th element .in trans supply of transposase protein [The family of mobile DNA elements also include non-autonomous Miniature inverted-repeat transposable elements (MITEs), whose transposition relies on an  protein . Sharing protein ,77. ThesWith common mechanisms of transposition and supposedly similar modes of regulation cut-and-paste DNA transposons embark on an evolutionary journey. Following invasion of a new host the newly arrived DNA transposon must proliferate and spread in the host population. After establishment and initial spreading, however, host- and self-regulation starts limiting transposition and inactivating transposons, eventually hampering mobilization of the element in the host. Without being able to transpose and proliferate, the inactive transposons are left as relics in the host genome and may eventually be lost by genetic drift.P elements and Tc1/mariners have spawned the strongest and most numerous horizontal transfer documentations , has bens  DNA transposable elements in maize, Chen et al. suggested a replicative mode of transposition [Ac/Ds transposition [Ac/Ds transposon severely inhibited transposition in a way that could be overcome by DNA replication. They revealed by in vitro studies of transposase binding to differently methylated Ac/Ds fragments that the effect of DNA replication relied on selective binding of the transposase to hemimethylated transposase binding sites. In fact, this selectivity was even shown to be specific for one of the two hemimethylated daughter elements produced by replication, making this element over six times as active. Though further mechanistic substantiation is needed, these data, supported by earlier studies on Ac/Ds transposition [In consideration of the abundance of DNA transposons and the cut-and-paste mechanism of transposition we are faced with an obvious question: how can elements, which move in a non-replicative way, ever increase in numbers within a genome? Based on genetic studies of the position . Accordiposition ,96. Specposition , Ros andposition ,96,97 anposition , provideTn10/IS10 [Tn5/IS50 [IS4 family [Ac/Ds elements. Hence, both elements transpose preferentially from hemimethylated DNA with a specific preference for one of the strands. Interestingly, these two elements are, unlike many other prokaryotic DNA transposons, restricted to non-replicative cut-and-paste transposition [dam (DNA adenine methylase) sites in other prokaryotic DNA transposons like IS1 and IS26 [Additional support for replication of transposons through jumping to unreplicated DNA is provided by early studies on prokaryotic cut-and-paste transposons. Results from the prokaryotic DNA transposons n10/IS10  and Tn5/Tn5/IS50 ,99 of th4 family  reveal tposition ,102. Thiand IS26 ,103,104.et al. of homolog-dependent high-frequency loss of the P element DNA transposon in Drosophila [et al. However, if the template is the sister chromatid, which was shown to be preferred in a study of P elements [mariner superfamily, indicating that this model could be universal [Another model for replication of transposable elements arose from studies by Engels osophila . The modelements , the traniversal ,108.Ac/Ds elements by incomplete repair after transposon excision [hAT superfamily of cut-and-paste DNA transposons.Synthesis-dependent strand annealing  has beeexcision , indicatP element [mariner Sleeping Beauty element [P elements indicated interaction between the P element inverted repeats and the Drosophila homolog of the mammalian protein Ku70 [Sleeping Beauty transposase and Ku70 [The bigger picture of repair after transposon excision also involves other mechanisms. In non-homologous end-joining  - are major competing or complementary pathways [Sleeping Beauty transposase-Ku70-interaction [Sleeping Beauty [Sleeping Beauty transposase halts cell cycle progression from G1 through interaction with the transcription factor Miz-1  forpathways , cell cypathways  may deteeraction , or by mg Beauty . Here itgulation -123, incgulation ). Herebyt repair ,126. Themariner elements which always integrate into a TA dinucleotide [piggyBac which always integrate into TTAA tetranucleotides [hAT superfamily and P elements, are more flexible and insert into 8-bp integration sequences that may vary in nucleotide composition [Along with genomic excision transposons are faced with the task of locating new sites in the genome to integrate into. The target site preference of transposons varies between the different transposon families, but common to most elements is that the target site sequence is duplicated upon integration, leaving the inserted transposon flanked by small stretches of identical sequences. Some transposon elements are very strict in their sequence choice, such as the Tc1/cleotide , and pigleotides . Other tposition ,128.Sleeping Beauty transposition in the mouse germ line it was observed that 27% of the transposition events had occurred within 200 kb of the donor site, and 75% of the transposition events were found to be on the same chromosome [P elements [Tol2 element [Ac/Ds elements [Sleeping Beauty insertion sites are widely distributed in the genome, when transposition occurs from a donor plasmid [piggyBac transposon seems to exhibit no or little local hopping [In addition to the primary target site sequence, several genomic features influence transposon insertion site preference. One feature is the genomic distance from the transposon donor site. In a study of romosome . This phromosome , P elemeelements , the Tol element , and Ac/elements . As Slee plasmid ,51, loca hopping ,135, sugmariner elements is DNA bendability. Thus, the flexibility and overall topology may influence the access to the DNA at a given genomic position. Analyses of insertion sites of Tc1, Tc3, Himar1, Sleepng Beauty, and Minos transposons have shown that TA sites in DNA regions with increased bendability are preferred compared to TA sites in more stiff DNA regions [mariner preference still remains to be elucidated, but a possible explanation could be that flexible DNA is more easily attacked by the transposase catalytic site during transposition, and/or that the transposase interacts with cellular proteins associated with bendable DNA. Based on studies demonstrating the sensitivity of Hsmar1 transposition to DNA topology, it was speculated that a certain topology of the targeted sequence enables the transposase to create mechanical strain at the active site by bending the DNA. This may allow structural changes during release of the mechanical strain, making re-excision of the inserted transposon less likely [Another genomic feature that has importance for insertion site preference of several Tc1/ regions ,136,137.s likely .Sleeping Beauty insertion sites from cultured HeLa and K562 cells [Sleeping Beauty transposon has a fairly random integration profile with no preference for or against genes. The P element, in contrast, has a strong preference for integrating near promoter regions, and analyses of more than 9,000 insertions in the Drosophila genome showed that 73% of P element insertions lie within 500 bp of an annotated 5′ transcription start site [Drosophila genome were targeted by an insertion [P element target sites and Origin Recognition Complex (ORC) binding sites at replication origins. ORC associates with open chromatin and promoters thus explaining the correlation between P element targets and promoters [Tol2 and piggyBac transposons are shown to exhibit nonrandom integration patterns. Of 113 Tol2 insertion sites in HeLa cells, 48% were found to be in transcriptional units and a significant number was observed to be close to transcriptional start sites [Tol2 in HEK293 cells [piggyBac transposon in HeLa and HEK293 cells [piggyBac has been observed to have a preference for integrating into active genes in primary human T cells [Retroviruses have been shown to have a non-random integration profile with some genomic locations being favored over others. Lentiviruses, for instance, prefer to integrate into actively transcribed units, while gammaretroviruses have a preference for integrating near the 5′ end of actively transcribed genes . Among t62 cells ,139, mou62 cells , NIH 3 T62 cells , and hum62 cells , have reart site ,142. A rnsertion . Furtherromoters . Also thrt sites , a prefe93 cells  and in p93 cells . Similar93 cells ,63,146, 93 cells , and pri93 cells ,147. In  T cells ). LEDGF/In a likely scenario in nature, transposon colonization of the germline of a species is initialized by horizontal transmission between species followed by dramatic multiplication and spread of the mobile DNA element. Over time, the copy number will level out at a steady-state level as the result of the co-evolution of host and transposon. The delicate balance between continuous spread and proliferation of active elements and a decline in host fitness caused by transposon-induced damage to the genome results in selection at the host level against high transposon activity. It also seems reasonable that transposons on this leg of their evolutionary journey travel with regulatory mechanisms that may directly influence the transposition rate and reduce the genetic harm to the host. Although such control mechanisms are vaguely characterized, there is evidence that transposons not only are regulated by host defense pathways, including transcriptional silencing and posttranscriptional silencing by RNA interference (see later), but also that the elements themselves possess autoregulatory control mechanisms with the transposase protein as the key player.mariner and other transposon superfamilies is inhibited by elevated levels of transposase, indicating, hence, that transposon activity during evolution has been regulated by self-inhibitory regulatory mechanisms. Events of transposase-based autoregulation, collectively referred to as overproduction inhibition (OPI), were first described for eukaryotic transposons by Lohe and Hartl [in vitro transposition studies including purified transposase protein derived from the mobile prokaryotic element Tn10[mariner transposon present in the genetically engineered white-peach gene of Drosophila mauritiana, Lohe and Hartl found reduced levels of mariner excision as a result of increasing transposase dosage. Hence, homozygous flies carrying two Mos1 transposase-expressing alleles or flies carrying copies of the transposase expression cassette on two different chromosomes showed significantly lower excision rates than flies with a single cassette [Mos1 excision was further reduced by heat-shock treatment of flies containing transposase genes driven by a chimeric heat-shock hsp70-derived promoter, resulting in excision rates that were reduced more than 50% in comparison to untreated flies carrying a single transposase gene [Mos1 impairs paired-end complex assembly in a mobility shift assay, increasing dosages of purified Mos1 transposase expressed in Escherichia coli were found not to cause OPI in an in vitro transposition assay [Himar1 mariner element was found to induce strong OPI even at low concentrations [Himar1 transposase-expressing plasmid result in reduced transposition rates [In attempts to mimic DNA transposition in nature most studies of DNA transposons with activity in eukaryotes rely on genetically engineered transposons and cloned versions of transposases that have been genetically modified to obtain activity. In addition, in functional studies transposase expression is often driven by promoters that facilitate high levels of transposase production. Despite potential discrepancies between actual mechanisms that are in play during genome evolution and in short-term laboratory assays, there is solid evidence that DNA transposition of members of the Tc1/nd Hartl , but hadment Tn10. In trancassette ,151. In ase gene ,152,153.on assay ,155. In trations ,154. Traon rates .Himar1 element in human cells [Sleeping Beauty transposase has attracted special attention due to its high activity in human cells [Sleeping Beauty transposase from a heat-inducible promoter and from two strong constitutive promoters, Izsvak and coworkers were not able to detect any negative regulatory effects of high transposase production levels in cultured HeLa cells. Hence, within the studied levels of transposase (as detected by western blotting) the transposition rates did not plateau or decrease with increasing transposase expression. [Sleeping Beauty transposition rate declined only moderately (about 1.5-fold) [in vitro-documented OPI effects of Tc1/mariner elements may be influenced by factors like cell type and transfection rate.Despite recent evidence for activity of the an cells , the funan cells . By tranression. . Howeverression. . In thes.5-fold) , indicatpiggyBac, and optimized derivatives of this system, may transpose in human cells with efficiencies that are comparable to improved versions of the Sleeping Beauty transposon. Although a single study shows OPI of piggyBac in HEK-293 cells [mariner transposases are generally less sensitive to OPI [Sleeping Beauty, piggyBac and Tol2, the hyperactive SB100X transposase was shown to be more prone to regulation by OPI, reaching optimum transposition conditions at a 1:10 transposase:transposon ratio [piggyBac transposase-to-transposon ratios as high as 43:1 did not induce OPI in HEK-293 cells, whereas a similar ratio for Sleeping Beauty caused OPI [Based on many reports from the last years, it appears now to be generally accepted that 93 cells , it appee to OPI ,63,157. on ratio . Notablyin vivo use of transposable elements. However, the question remains if negative dosage effects observed in transient transfection studies reflect true biological mechanisms with impact on natural populations and the regulation of DNA transposition through evolution or, perhaps less excitingly, is the result of artificial overproduction of an enzyme with toxic effects on the treated cells. One can easily envisage putative evolutionary implications of OPI as one of many possible ways of regulating genomic transposition. In a likely scenario, low levels of transposase expressed from a relatively low number of integrated elements may provide optimal conditions for mobility. However, as the number of active elements increases over time the overall transposase dosage may reach a certain threshold beyond which transposition is reduced. As of today the mechanisms involved are unknown but may possibly include posttranslational interactions between transposase molecules. Recent studies of a resurrected Hsmar1 mariner transposon support a model by which transposon activity is autoregulated through competition between transposase subunits for binding sites within existing transposon elements [Sleeping Beauty study this was found not to be the case [OPI-based regulation of transposition represents an additional layer of complexity in transposon-based gene transfer and may have crucial influence on the elements . In accoelements . In anotthe case .Eukaryotic genomes consist of regions of transcriptionally active euchromatin and transcriptionally inactive heterochromatin. The organization of genomes into such regions is controlled by an interplay between DNA methylation and histone modifications, and these epigenetic modifications have been shown to be closely associated with the transcriptional state of many eukaryotic genes ,160.Analyses of DNA methylation patterns in both plant and animal species have revealed that endogenous transposon elements are heavily methylated . Recent Arabidopsis thaliana mutated in the DDM1 (decrease in DNA methylation) gene revealed increased transposition rates [et al. who, by microarray and chromatin immunoprecipitation (ChIP) studies in A. thaliana, showed that heterochromatic CACTA transposons and gypsy-like retrotransposons are activated in DDM1 gene mutants [A. thaliana was enhanced significantly in plants defective in either CG or non-CG DNA methylation[IAP retrotransposons was also observed in mouse embryos deficient of a fully functional Dnmt1 (DNA methyltransferase-1) gene [Studies of transposition in on rates ,165. Sim mutants . These r mutants . Neverththylation. Althoug-1) gene .Sleeping Beauty transposition is enhanced by transposon DNA methylation, suggesting that transposon mobilization is supported by CpG methylation [Sleeping Beauty and Frog Prince elements, leading to the assumption that methylation and a resulting tight chromatin structure is beneficial for transposition of these particular elements [Sleeping Beauty transposon insertion, excision efficiencies were observed to be 100-fold higher when the Sleeping Beauty transposon was in a heterochromatic conformation compared to control clones without the heterochromatic conformation [Sleeping Beauty transposon in mouse embryonic stem cells was also observed in a study in which transposition efficiency of the piggyBac transposon at the same time was shown to be reduced upon methylation [Sleeping Beauty element appears to have an affinity for heterochromatic regions [As an intriguing exception to these findings, it appears that hylation . Recent elements . In mousormation . Improvehylation . In acco regions . It is tTol1 and Tol2 transposable elements, both members of the hAT superfamily, are unique among the DNA transposons, as they are the only natural active elements to have been discovered so far in vertebrate genomes. The Tol2 element has had a rapid expansion in the genome of its host, the medaka fish, in the past, but a high spontaneous transposition rate is not observed in the genome of current laboratory fish strains, suggesting that Tol2 has already reached a steady-state level where the transposition frequency is controlled by host mechanisms [Tol2 transposition. Hence, in a study by Iida et al. [Tol2 transposon did not inhibit transposition by transcriptional silencing of the transposase gene. In studies involving other hAT elements, methylation at the ends of the plant transposon Tam3 (from Antirrhinum) was shown to cause repression of transposition [Ac/Ds element was shown to inhibit Ac transposase binding [hAT transposon superfamily is due to inhibited transposase binding to transposon binding sites.The chanisms . Evidenca et al.  medaka fposition , and DNA binding . These fSleeping Beauty element carrying a transgene, and not the natural transposase gene, was analyzed in transgenic mice containing single-copy transposon insertions [Sleeping Beauty transposons inserted into the mouse genome were heavily methylated in the ROSA26 promoter and the eGFP coding sequence. In contrast, the endogenous mouse ROSA26 promoter was devoid of CpG methylation in the transgenic mice. It appears that the host cells were able to distinguish between the endogenous genomic sequence and the exogenous counterpart inserted by Sleeping Beauty, suggesting that the transposon was specifically recognized and targeted by the host cellular epigenetic modification system. In another study, however, Sleeping Beauty transgenic mice containing a transgene cassette comprised of a human K14 promoter and the Agouti reporter gene did not show significant levels of CpG methylation at the inserted transgene [Sleeping Beauty cargo DNA sequences rather than transposon inverted repeat sequences influence CpG methylation status. We have at several occasions observed transcriptional down-regulation and silencing of Sleeping Beauty-inserted transgene cassettes [piggyBac was less vulnerable to silencing than a cassette delivered by the Sleeping Beauty counterpart, lending support to the notion that the level of silencing was influenced by the distinct integration profiles [Rather than affecting the process of transposition itself, epigenetic modifications may impact transposase expression and thereby indirectly regulate transposition activity. CpG methylation of the sertions . The DNAransgene , indicatassettes -179, butLINE-1 and IAP retrotransposons in 2-cell embryos. A subsequent loss of expression in 8-cell embryos was shown to correlate with a loss of the activating trimethylation of H3K4 rather than gain of the repressive trimethylation of H3K9 [The role of histone modifications in transposon control is under continuous investigation. A recent study in murine embryos showed reactivation of the  of H3K9 . It is nTo produce transposase proteins that facilitate jumping of transposons to a new location in the genome the transposon open reading frame must first be copied into mRNA. The evolution of host silencing mechanisms, based on the ability of small inhibitory RNA molecules to guide mRNA degradation in a sequence-specific fashion, has allowed higher organisms to defend the genome against mobile elements and harmful effects of insertional mutagenesis and genomic instability. Formation of transposon-derived double-stranded RNA (dsRNA) is a hallmark of RNAi-based suppression of transposition. Figure C. elegans, transcription of Tc1 sequences is initiated in the flanking sequences and not within the inverted repeats [Tc1 transposition in wildtype worms occurs only in somatic cells and not in the germ line, identification of mutants with active transposition in germ cells has led to identification of genes that play an active role in the RNAi response in C. elegans[rde-2 and mut-7 among others, encode conserved proteins involved in degrading RNA by RNAi. In agreement with data showing that mutations in these genes relieve a block on transposition [Tc1 are much less abundant in mutant worms defective in both RNAi and transposon silencing [The lack of easy identifiable regulatory elements in DNA transposons has fueled the idea that expression of the transposase gene in original elements depends on endogenous regulatory sequences flanking the inserted transposon. This may give rise to read-through RNA transcripts that may serve as templates for transposase synthesis. In  repeats . Interes repeats . As Tc1 . elegans,183. Witposition ,183, smailencing .Drosophila germline, ra-siRNA-directed silencing serves to suppress transposon expression, but through a yet incompletely understood RNA processing pathway that does not involve siRNA production by Dicer. Such ra-siRNA effectors are referred to also as piRNAs due to their association with the Argonaute subfamily of PIWI proteins [Drosophila PIWI mutants.Fastly accumulating evidence suggests that retrotransposons in wide variety of host organisms are suppressed through the RNAi pathway. In mouse oocytes, repeat-associated siRNAs (ra-siRNAs) derived from retroelements have been identified and are likely effectors in an anti-transposon response through RNAi , as suppproteins ,188 and proteins ,190. Intproteins ,192 and proteins . FurtherLINE-1 retroelements comprise about 17% of the human genome and an estimated 100 of these elements are fully capable of transposition. Sense and antisense promoters in the 5′ untranslated region of LINE-1 direct production of bi-directional RNA transcripts which can potentially anneal to form double-stranded RNA  elements cannot distinguish autonomous from non-autonomous elements in choosing its substrate  accumulation of mutations in the transposase gene or (ii) more severe genetic alterations, like deletions, caused by vertical inactivation. Among Tc1-like elements in fish the salmonid subfamily appears to be the youngest and most recently active [Sleeping Beauty transposase have been developed by mutagenesis, leading to several versions of the transposase with enhanced transposition properties [Sleeping Beauty transposase  under certain experimental conditions [piggyBac transposition have been achieved by codon optimization of the transposase gene, leading to induced levels of expression [piggyBac transposase (HyPBase) was recently identified by screening of a transposase mutant library and combining beneficial mutations in a single transposase variant [in vivo model, SB100X and HyPBase have been found to increase in vivo efficacy above what has been observed with previous transposase variants [Despite the proven potency of smission . Hence, y active . Based ooperties ,136,203.nditions . Moreovepression ,206. Als variant . By emplvariants ,53 and cin vitro-transcribed mRNA may serve as a rich source of transposase [Conventional DNA transposon systems consist of two plasmids, one helper plasmid carrying the transposase expression cassette and one donor plasmid carrying the DNA transposon Figure , panel ansposase , eliminaSleeping Beauty transposon system was first demonstrated in mouse liver with adenoviral vectors [Sleeping Beauty system [Sleeping Beauty DNA transposition from integrase-defective lentiviral vectors (IDLVs)  Figure , panel bansgenes ,204,213.ansgenes . Most reansgenes . Effortsansgenes ,216,217.Sleeping Beauty is by far the most studied for gene transfer purposes. Several studies have demonstrated high levels of gene insertion in mouse liver after hydrodynamic injection of naked DNA into the tail vein. Proof-of-principle was initially provided by Yant et al. who obtained persistent levels of human coagulation factor IX (hFIX) after hydrodynamic injection of plasmids carrying the transgene-tagged transposon and the transposase expression cassette, respectively [Sleeping Beauty system has been successful in liver-directed treatment of mucopolysaccharidosis types I and VII [in vitro synthesized RNA encoding the Sleeping Beauty transposase, as opposed to plasmid DNA, is sufficient to catalyze vector transposition in mouse liver [Sleeping Beauty-directed transfer of hFIX showed efficacy but also demonstrated severe OPI in mice treated with too high levels of plasmid DNA with transposase expression driven by a standard cytomegalovirus (CMV) promoter. Hence, whereas a single microgram of transposase-encoding plasmid DNA was found to facilitate high levels of transposition, resulting in correction of the disease in mouse model of hemophilia, injection of 25 μg transposase-encoding plasmid DNA on the other hand did not result in DNA transposition [Sleeping Beauty, or rather is caused by toxicity in hepatocytes with high expression of the transposase. In either case, these findings marked the importance of recognizing specific characteristics of the vector technology and balancing the expression of the transposase for each specific application.Among the elements with activity in mammalian cells ectively . Deliverectively . As thisectively , recent ectively . In addi and VII  and tyro and VII , and admse liver ,220. Iniposition . It is nSleeping Beauty-containing plasmid vectors have been delivered to lung epithelial cells [Sleeping Beauty-treated mouse lungs may last as long as 3 months after injection [Sleeping Beauty have been elegantly demonstrated in human glioblastoma xenografts in mice in which tumor-induced angiogenesis was inhibited by Sleeping Beauty-based co-expression of soluble vascular endothelial growth factor and a angiostatin-endostatin fusion variant [Sleeping Beauty transposase was present, allowing persistent expression of the two genes.By administration of DNA-polyethylenimine complexes, al cells , allowinal cells . Gene exnjection . Promisi variant ,19. SustSleeping Beauty element was re-awakened and soon after adapted for in vivo gene delivery facilitating persistent gene expression. Despite the various examples of pre-clinical efficacy for DNA transposon-based in vivo gene therapy, the road to the clinic will wind through additional experimentation and evidence of therapeutic effects in large animal models. As we keep learning more about the properties and details of the different transposable elements and continue developing both nonviral and viral delivery technologies, in vivo applicability could be waiting around the next corner. Until then, the history of mobile elements is helping us refining these engineering tools even further for use in both biomedical experimentation and clinical settings. With the current primary focus on the Sleeping Beauty and piggyBac elements, mechanisms that guide the distinct integration patterns, OPI, gene cargo capacities, putative cell type variations, and sensitivity to epigenetic silencing of these two elements seem to be key focus points for optimizing the performance of these elements further.Fifteen years have gone since the ex vivo therapies. Transposon-based gene insertion in plasmid-transfected primary T cells [ex vivo genetic treatment. In fact, the first clinical trial based on transposon-directed gene integration aims at inserting a gene encoding a chimeric antigen receptor specific for CD19 in primary T cells [The combination of improved transposons, possibly mobilized from DNA minicircles free of bacterial sequences , and eng T cells -225, hem T cells ,226-228, T cells ,230 is c T cells  and has  T cells -233. ThrThe authors declare that they have no competing interests.The manuscript was prepared by KAS, PRA, NS and JGM. All authors read and approved the final manuscript.


Psychotic experiences occur at a much greater prevalence in the population than psychotic disorders. There has been little research to date, however, on genetic risk for this extended psychosis phenotype. We examined whether COMT or BDNF genotypes were associated with psychotic experiences or interacted with childhood trauma in predicting psychotic experiences.Psychiatric interviews and genotyping for COMT-Val158Met and BDNF-Val66Met were carried out on two population-based samples of 237 individuals aged 11-15 years. Logistic regression was used to examine for main effects by genotype and childhood trauma, controlling for important covariates. This was then compared to a model with a term for interaction between genotype and childhood trauma. Where a possible interaction was detected, this was further explored in stratified analyses.While childhood trauma showed a borderline association with psychotic experiences, COMT-Val158Met and BDNF-Val66Met genotypes were not directly associated with psychotic experiences in the population. Testing for gene x environment interaction was borderline significant in the case of COMT-Val158Met with individuals with the COMT-Val158Met Val-Val genotype, who had been exposed to childhood trauma borderline significantly more likely to report psychotic experiences than those with Val-Met or Met-Met genotypes. There was no similar interaction by BDNF-Val66Met genotype.The COMT-Val158Met Val-Val genotype may be a genetic moderator of risk for psychotic experiences in individuals exposed to childhood traumatic experiences. Much research has established that psychotic experiences have a higher prevalence in the general population than psychotic disorders -4. A metWe previously suggested that genes for psychosis may, in fact, be genes for the broader ‘extended psychosis phenotype’, made up not just of individuals with psychotic disorders but also a much larger population of individuals with psychotic experiences . InvestiTwo frequently studied genetic variants in psychosis research are the catechol-o-methyl-transferase (COMT) Val158Met and the brain-derived neurotrophic factor (BDNF) Val66Met polymorphisms -32. COMTThe present study aimed to explore the role of the COMT-Val158Met and BDNF-Val66Met polymorphisms in two community samples with psychotic experiences. We wished to test for a direct association between these polymorphisms and psychotic experiences in the general population. Furthermore, we aimed to test a gene-environment interaction, specifically, that those with the COMT-Val158Met Val-Val genotype or the BDNF-Val66Met Met-Met/Val-Met genotypes are more susceptible to psychotic experiences following trauma than those with other genotypes.The sample consisted of 237 adolescents from two independent studies: 123 participants from the Adolescent Brain Development (ABD) study and 114 participants from the Challenging Times (CT) study. The study methodologies have been previously reported -48. DetaBriefly, the ABD study took place in counties Dublin and Kildare, Ireland, which contains a mixture of urban and suburban housing types of different socioeconomic status. Out of twenty seven schools approached, sixteen agreed to participate (59.3%), while four said they may participate at another time and seven (25.9%) declined participation. Participants were aged 11-13 years and were in the two most senior classes in the Irish primary school system. Written parental consent was required for adolescents to take part and 1,131 signed consent forms were returned out of 2,190 distributed (52%). Of the 1,131 adolescents who took part in the survey study, 656 (58%) indicated an interest in participating in the interview study and a random sample of 212 of these attended for interview. Those who participated at interview did not differ from those who did not on levels of psychopathology as determined by the SDQ. Those who attended for interview were also representative of the Irish population in terms of socioeconomic status and ethnic background. Among this sample, a total of 168 individuals provided genetic samples. There was insufficient DNA for analysis in 45 of these samples, resulting in sufficient DNA for genotyping the BDNF-Val66Met or COMT-Val158Met polymorphisms in 123 participants. The CT study took place in the geographical catchment area of a child and adolescent mental health team in north Dublin, containing a population of 137,000. This urban area includes pockets of severe inner city deprivation, large suburban housing estates and more affluent areas of private housing. The population includes a higher proportion from lower socioeconomic backgrounds compared to the overall Irish population. A sample of 12 secondary schools in the catchment area was selected using stratified random sampling according to the approximate socio-economic status of the school. Of these 12 schools, eight participated in the study. One school did not participate owing to concerns that questioning pupils about suicidal thoughts might be harmful and three schools that agreed to participate were excluded because insufficient parental consent forms were received <50%). The exclusion of these schools did not skew the socio-economic distribution of the remaining schools. A total of 743 students aged 12-15 years in eight schools were screened for psychopathology using the Strengths and Difficulties Questionnaire , which m%. The exIn total, between both studies, there was sufficient DNA from 226 participants for analysis of COMT-Val158Met (115 from ABD study and 111 from CT study) and 222 participants for analysis of BDNF-Val66Met (116 from ABD study and 106 from CT study). This data is not available online owing to the necessity to protect the confidentiality of the young people who participated (and discussed the sensitive areas of psychotic experiences and history of abuse). However, we are happy to facilitate requests from researchers to freely review the data.www.hapmap.org) were genotyped for each SNP (rs4680 and rs6265) for quality control purposes and were found to be 100% concordant with available HapMap data for these SNPs.Genetic analysis was carried out using DNA extracted from saliva samples.  The COMT (rs4680) and BDNF (rs6265) SNPs were genotyped using Taqman® SNP genotyping assays on a 7900HT sequence detection system (Applied Biosystems).  The call rate for the Taqman genotyping was >95% and the samples were in Hardy-Weinberg equilibrium (p>0.05).  Along with these samples, thirteen HapMap CEU DNA samples (Participants and their parents were interviewed using the Schedule for Affective Disorders and Schizophrenia for School-aged Children (K-SADS), Present and Lifetime versions. The K-SADS is a well-validated semi-structured research diagnostic interview for the assessment of Axis I psychiatric disorders in children and adolescents . ChildreEthical approval for the Adolescent Brain Development study was received from Beaumont Hospital medical ethics committee and for the Challenging Times study from the Mater Misericordiae University Hospital medical ethics committee. Informed assent was obtained from participants under age 16 years with informed consent received from their parents. In addition, all individual data was anonymised with no identifying characteristics present. Written informed consent was obtained from the parent or guardian of participants.Differences between those who participated in interview and provided genetic samples and those who participated but did not were assessed using chi-squared tests. Similarly, differences between those with versus without psychotic experiences were assessed using chi-squared tests or Fisher’s exact test as appropriate. Based on the hypothesis that COMT-Val158Met genotype can moderate the influence of childhood trauma on psychotic experiences, we tested whether psychotic experiences would be predicted by an interaction between a gene  and an environment (childhood trauma). To analyse the interaction between childhood trauma and genotypes , logistic regression was used, with the absence or presence of psychotic experiences as the dependent variable. Sex, school grade and cannabis use were used as covariates. School grade was included as a categorical variable with four levels, one for each grade. The first two grades were recruited for the ABD study and the second two grades for the CT study. This variable therefore approximated the effects of both age and study group. Cannabis use and sex were also included to control for potential confounding by these factors. A logistic regression model was first fitted to determine the main effects of childhood trauma and COMT-Val158Met genotype (model 1). Second, to explore the interaction effect of COMT-Val158Met and childhood trauma, a second model was fitted with an interaction term (model 2) and this was compared to the first model using the likelihood ratio test. This process was also repeated separately in the same manner to examine the main effect of BDNF-Val66Met genotype and interaction between this genotype and childhood trauma. Finally, where these tests indicated an important G X E interaction, the effect of this interaction was explored within each genotype group, using stratified tables with odds ratios and 95% confidence intervals. Statistical analyses were conducted using Stata version 11.0 for Windows. All statistical tests were two-sided; P-values less than 0.05 were judged statistically significant.Those who attended for interview and provided usable genetic samples were broadly similar to those who attended for clinical interview but did not provide usable genetic samples  of the total sample. Childhood trauma was reported by 21/237 (9%). This included 13 (5.5%) who witnessed domestic violence, 9 (3.8%) who described child physical abuse and 4 (1.7%) who described child sexual abuse. There was overlap in these individuals, with some experiencing multiple forms of childhood trauma. 2<0.01, P=0.958). Following this, the effect of childhood trauma on psychotic experiences was examined in each COMT-genotype sub-group see . ChildhoThe current study provides, to our knowledge, the first evidence of a gene-environment interaction between the COMT-Val158Met genotype and childhood traumatic experiences in terms of risk for clinically assessed psychotic experiences in the population. When stratified by COMT-Val158Met genotype, individuals with the Val-Val genotype who experienced childhood trauma were borderline significantly more likely to report psychotic experiences compared to adolescents with the Val/Met and Met/Met genotypes. Our findings should be viewed in the context of advances in understanding of the genetic underpinnings of the extended psychosis phenotype. Binbay et al examined familial risk for this extended phenotype, which ranges from psychotic experiences without impairment to clinical psychotic disorder. They suggested that there are high prevalence and low prevalence genetic risks, interacting with environmental risks, operating across the extended psychosis phenotype but particularly in those with clinical psychotic disorders . In thisOur results suggesting a possible interaction between the COMT-Val158Met Val-Val genotype and childhood trauma in association with psychotic experiences are consistent with previous findings regarding gene-environment interactions between COMT-Val158Met and stressful/traumatic experiences in association with the extended psychosis phenotype. Consistent with our findings, the Val allele was associated with self-reported psychotic symptoms interacting with cannabis use in an adult Dutch population sample  and withIt has become increasingly clear that psychotic experiences at this age mark risk for a wide range of psychopathology, not limited to psychotic disorders ,2,11,55.The molecular mechanisms of gene-environment interactions remain obscure. Childhood trauma-dependent DNA-demethylation has been associated with increased stress-dependent gene transcription and long-term dysregulation of the stress hormone system in the case of FK506 binding protein 5, a regulator of the stress hormone system . ChildhoWe found no association between BDNF genotype and psychotic experiences. Furthermore, we found no gene-environment interaction between BDNF genotype and childhood trauma in predicting psychotic experiences. This differs from the findings of Alemany and colleagues , who fouAmong the strengths of this study is the use of gold-standard clinical interviews of both parent and child to ascertain psychotic experiences and childhood trauma. Most studies on psychosis and childhood trauma have been conducted with adults, meaning there is a considerable temporal gap between events and symptom assessment, increasing the risk of recall bias. Given that our research was conducted in youths, however, the assessment of psychotic experiences took place relatively soon after the time of the traumatic events, reducing recall bias risk. A further strength of this study is that we have controlled for important potential confounders to a gene environment interaction, particularly cannabis use . A number of limitations must be considered in evaluating the results of this study. Firstly, psychotic symptoms and trauma history were determined simultaneously, meaning that we cannot be certain that childhood trauma preceded psychotic experiences. Furthermore, the timing of trauma was not clearly ascertained. Recent findings suggest that the trauma is likely to be a causal risk factor for psychotic experiences but that this relationship is also partly bi-directional . HoweverOur findings provide evidence in support of a gene-environment interaction between the COMT-Val158Met polymorphism and traumatic experiences in terms of risk for psychotic experiences in the population. This adds to the existing literature suggesting increased paranoia , schizot