In vitro antimicrobial activity of equine platelet lysate and mesenchymal stromal cells against common clinical pathogens.

Septic arthritis is considered a medical emergency. Disease following bacterial colonization can lead to significant morbidity and mortality and requires costly treatment. Antimicrobial properties of regenerative therapies, including mesenchymal stromal cells and platelet products, have been researched extensively in human medicine. Although fewer studies have been conducted in veterinary species, they have shown promising results. The purpose of this study was to evaluate bacterial suppression by equine platelet lysate (EPL) and adipose-derived mesenchymal stromal cells (ASCs) in vitro. We hypothesized that both products would significantly inhibit the growth of Staphylococcus aureus and Escherichia coli. Pooled blood from 10 horses was used for production of EPL. Mesenchymal stromal cells were isolated from adipose tissue harvested from the gluteal region of 3 horses. The study evaluated 3 treatment groups: 10 × EPL, 1.6 million ASCs, and a control, using an incomplete unbalanced block design with repeated measurements. Optical density readings and colony-forming units/mL were calculated at 0, 3, 6, 9, 12, 18, and 24 hours. Decreased bacterial growth was seen at multiple time points for the S. aureus-ASC and S. aureus-EPL treatments, supporting our hypothesis. Increased bacterial growth was noticed in the E. coli-EPL group, with no difference in the E. coli-ASC treatment, which opposed our hypothesis. A clear conclusion of antimicrobial effects of EPL and ASCs cannot be made from this in vitro study. Although it appears that ASCs have a significant effect on decreasing the growth of S. aureus, further studies are needed to explore these effects, particularly in Gram-positive bacteria.

L'arthrite septique est considérée comme une urgence médicale. La maladie consécutive à une colonisation bactérienne peut entraîner une morbidité et une mortalité importantes et nécessite un traitement coûteux. Les propriétés antimicrobiennes des thérapies régénératives, y compris les cellules stromales mésenchymateuses et les produits plaquettaires, ont fait l'objet de recherches approfondies en médecine humaine. Bien que moins d'études aient été menées chez les espèces animales, elles ont montré des résultats prometteurs. Le but de cette étude était d'évaluer la suppression bactérienne par le lysat plaquettaire équin (EPL) et les cellules stromales mésenchymateuses adipeuses (ASC) i n vitro. Nous avons émis l'hypothèse que les deux produits inhiberaient de manière significative la croissance de Staphylococcus aureus et d'Escherichia coli. Un pool de sang de 10 chevaux a été utilisé pour la production d'EPL. Des cellules stromales mésenchymateuses ont été isolées à partir de tissu adipeux prélevé dans la région fessière de trois chevaux. L'étude a évalué trois groupes de traitement : 10 × EPL, 1,6 million d'ASC et un témoin, en utilisant un design en blocs non équilibrés incomplets avec des mesures répétées. Les lectures de densité optique et les unités formatrices de colonie/mL ont été calculées à 0, 3, 6, 9, 12, 18 et 24 heures. Une diminution de la croissance bactérienne a été observée à plusieurs moments pour les traitements S. aureus-ASC et S. aureus-EPL, soutenant notre hypothèse. Une croissance bactérienne accrue a été remarquée dans le groupe E. coli-EPL, sans différence dans le traitement E. coli-ASC, ce qui s'opposait à notre hypothèse. Une conclusion claire des effets antimicrobiens de l'EPL et des ASC ne peut pas être tirée de cette étude in vitro. Bien qu'il semble que les ASC aient un effet significatif sur la diminution de la croissance de S. aureus, d'autres études sont nécessaires pour explorer ces effets, en particulier chez les bactéries à Gram positif.(Traduit par Docteur Serge Messier).

Publisher Correction: Evidence of radial Weibel instability in relativistic intensity laser-plasma interactions inside a sub-micron thick liquid target.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evidence of radial Weibel instability in relativistic intensity laser-plasma interactions inside a sub-micron thick liquid target.

Super-intense laser plasma interaction has shown great promise as a platform for next generation particle accelerators and sources for electron, x-rays, ions and neutrons. In particular, when a relativistic intense laser focus interacts with a thin solid density target, ionized electrons are accelerated to near the speed of light (c) within an optical cycle and are pushed in the forward and transverse directions away from focus, carrying a significant portion of the laser energy. These relativistic electrons are effectively collisionless, and their interactions with the ions and surrounding cold electrons are predominantly mediated by collective electromagnetic effects of the resulting currents and charge separation. Thus, a deeper understanding of subsequent high energy ions generated from various mechanisms and their optimization requires knowledge of the relativistic electron dynamics and the fields they produce. In addition to producing MV/m quasi-static fields, accelerating the ions and confining the majority of the electrons near the bulk of the laser target, these relativistic electron currents are subject to plasma instabilities like the Weibel instability as they propagate through the thermal population in the bulk target. In this work, we present high temporal (100 fs) and spatial (1 µm) resolution shadowgraphy video capturing relativistic radial ionization front expansion and the appearance of filamentation radiating from the laser spot within a sub-micron thick liquid sheet target. Filamentation within the region persists for several picoseconds and seeds the eventual recombination and heating dynamics on the nanosecond timescale. A large scale three-dimensional particle-in-cell (PIC) simulation of the interaction revealed the presence of strong magnetic fields characteristic of Weibel Instability, and corroborated the relativistic radial expansion of the ionization front, whose speed was determined to be 0.77c. Both the experimental and simulation results strongly point towards the target field ionization and the outward expanding hot electron current as the cause of the radial expansion.

Soma-germ line interactions and a role for muscle in the regulation of C. elegans sperm motility.

The development of highly differentiated sperm cells that are specialized for navigating to and fusing with an oocyte is essential for sexual reproduction. As a major part of differentiation, sperm undergo extensive post-meiotic maturation en route to the oocyte. This is regulated largely by soma-derived cues. In Caenorhabditiselegans, this process is called sperm activation, and it transforms immotile spermatids into migratory fertilization-competent cells. Here, we show that the negative regulator of sperm activation, SWM-1, is produced in an unexpected cell type: body wall muscle. SWM-1 is secreted into the body cavity and enters the gonad; there, it is present with its likely target, TRY-5, a spermiogenesis activator. We show that, in addition to SWM-1, the somatic gonad and body fluid can exchange other factors, suggesting that soma-germ line transfer could affect other reproductive processes. In addition, we show that SWM-1 may have a separate role in the sperm migratory environment, to which it is contributed by both males and hermaphrodites. These findings reveal that late stages in gamete differentiation can be regulated at the whole-organism level by broadly secreted factors.This article has an associated 'The people behind the papers' interview.

Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

The formation of an abdominal aortic aneurysm (AAA) is characterized by inflammation, macrophage infiltration, and vascular remodeling. In this study, we tested the hypothesis that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) immunomodulate aortic inflammation, to mitigate AAA formation via modulation of microRNA-147. An elastase-treatment model of AAA was used in male C57BL/6 wild-type (WT) mice. Administration of EVs in elastase-treated WT mice caused a significant attenuation of aortic diameter and mitigated proinflammatory cytokines, inflammatory cell infiltration, an increase in smooth muscle cell α-actin expression, and a decrease in elastic fiber disruption, compared with untreated mice. A 10-fold up-regulation of microRNA (miR)-147, a key mediator of macrophage inflammatory responses, was observed in murine aortic tissue in elastase-treated mice compared with controls on d 14. EVs derived from MSCs transfected with miR-147 mimic, but not with miR-147 inhibitor, attenuated aortic diameter, inflammation, and leukocyte infiltration in elastase-treated mice. In vitro studies of human aortic tissue explants and murine-derived CD11b+ macrophages induced proinflammatory cytokines after elastase treatment, and the expression was attenuated by cocultures with EVs transfected with miR-147 mimic, but not with miR-147 inhibitor. Thus, our findings define a critical role of MSC-derived EVs in attenuation of aortic inflammation and macrophage activation via miR-147 during AAA formation.-Spinosa, M., Lu, G., Su, G., Bontha, S. V., Gehrau, R., Salmon, M. D., Smith, J. R., Weiss, M. L., Mas, V. R., Upchurch, G. R., Sharma, A. K. Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

Relativistic electron acceleration by mJ-class kHz lasers normally incident on liquid targets.

We report observation of kHz-pulsed-laser-accelerated electron energies up to 3 MeV in the -klaser (backward) direction from a 3 mJ laser interacting at normal incidence with a solid density, flowing-liquid target. The electrons/MeV/s.r. >1 MeV recorded here using a mJ-class laser exceeds or equals that of prior super-ponderomotive electron studies employing lasers at lower repetition-rates and oblique incidence. Focal intensity of the 40-fs-duration laser is 1.5 · 1018 W cm-2, corresponding to only ∼80 keV electron ponderomotive energy. Varying laser intensity confirms electron energies in the laser-reflection direction well above what might be expected from ponderomotive scaling in normal-incidence laser-target geometry. This direct, normal-incidence energy spectrum measurement is made possible by modifying the final focusing off-axis-paraboloid (OAP) mirror with a central hole that allows electrons to pass, and restoring laser intensity through adaptive optics. A Lanex-based, optics-free high-acquisition rate (>100 Hz) magnetic electron-spectrometer was developed for this study to enable shot-to-shot statistical analysis and real-time feedback, which was leveraged in finding optimal pre-plasma conditions. 3D Particle-in-cell simulations of the interaction show qualitative super-ponderomotive spectral agreement with experiment. The demonstration of a high-repetition-rate, high-flux source containing >MeV electrons from a few-mJ, 40 fs laser and a simple liquid target encourages development of future ≥kHz-repetition, fs-duration electron-beam applications.

Mesenchymal Stem Cells Attenuate NADPH Oxidase-Dependent High Mobility Group Box 1 Production and Inhibit Abdominal Aortic Aneurysms.

OBJECTIVE: Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, smooth muscle activation, and matrix degradation. This study tests the hypothesis that macrophage-produced high mobility group box 1 (HMGB1) production is dependent on nicotinamide adenine dinucleotide phosphate oxidase (Nox2), which leads to increase in interleukin (IL)-17 production resulting in AAA formation and that treatment with human mesenchymal stem cells (MSCs) can attenuate this process thereby inhibiting AAA formation. APPROACH AND RESULTS: Human aortic tissue demonstrated a significant increase in HMGB1 expression in AAA patients when compared with controls. An elastase-perfusion model of AAA demonstrated a significant increase in HMGB1 production in C57BL/6 (wild-type [WT]) mice, which was attenuated by MSC treatment. Furthermore, anti-HMGB1 antibody treatment of WT mice attenuated AAA formation, IL-17 production, and immune cell infiltration when compared with elastase-perfused WT mice on day 14. Elastase-perfused Nox2(-/y) mice demonstrated a significant attenuation of HMGB1 and IL-17 production, cellular infiltration, matrix metalloproteinase activity, and AAA formation when compared with WT mice on day 14. In vitro studies showed that elastase-treated macrophages from WT mice, but not from Nox2(-/y) mice, produced HMGB1, which was attenuated by MSC treatment. The production of macrophage-dependent HMGB1 involved Nox2 activation and superoxide anion production, which was mitigated by MSC treatment. CONCLUSIONS: These results demonstrate that macrophage-produced HMGB1 leads to aortic inflammation and acts as a trigger for CD4(+) T-cell-produced IL-17 during AAA formation. HMGB1 release is dependent on Nox2 activation, which can be inhibited by MSCs leading to attenuation of proinflammatory cytokines, especially IL-17, and protection against AAA formation.

Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/ß-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures.

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/ß-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/ß-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.

Posterior quadrant epilepsy surgery: predictors of outcome.

PURPOSE: To identify predictors of seizure recurrence following posterior quadrant epilepsy surgery. METHODS: Between 1983 and 2008, 43 medically refractory epilepsy patients underwent posterior quadrant epilepsy surgery. Epilepsy surgery involved the occipital lobe in all cases; some cases also included resection of the adjacent parietal or temporal cortex. Using a logistic regression model, we evaluated the relationship between outcome (Engel class I-IV) and 5 outcome predictors: absence of a visual aura, a temporal lobe type aura, versive head movement unaccompanied by a visual aura, non-focal interictal scalp EEG, and surgical pathology other than low grade tumor or cortical dysplasia. We also determined the relative risk for significant post-operative cognitive decline of Wechsler intelligence test score among those receiving complete lobectomies compared to those receiving partial lobectomies. RESULTS: Overall, outcome was favorable at 1 year following surgery: 22 (51.2%) patients Engel class I, 10 (24%) patients Engel class II, 5 (12%) patients Engel class III, and 6 (14%) patients Engel class IV. The 3 best univariate predictors of seizure recurrence were versive head movement unaccompanied by visual aura, non-focal interictal scalp EEG, and pathology other than low grade tumor or cortical dysplasia. A multivariate predictor combining temporal lobe type aura, versive head movement unaccompanied by visual aura, non-focal interictal scalp EEG, and pathology other than low grade tumor or cortical dysplasia was optimum. Complete lobectomy significantly increased the risk of post-operative decline of Wechsler intelligence score. CONCLUSIONS: These findings indicate that posterior quadrant epilepsy surgery may provide sustained seizure control. A multivariate model combining temporal lobe type aura, versive head movement unaccompanied by a visual aura, non-focal interictal scalp EEG, and pathology other than low grade tumor or cortical dysplasia may contribute to predicting seizure recurrence following posterior quadrant epilepsy surgery. The extent of cortical resection may predict significant cognitive decline in post-operative Wechsler intelligence score.

Temporal pole proton preoperative magnetic resonance spectroscopy in patients undergoing surgery for mesial temporal sclerosis.

OBJECT: The purpose of this prospective study was to compare the results of proton MR spectroscopy (MRS) in temporal poles in patients with unilateral mesial temporal sclerosis (MTS) with the histopathological findings of the resected temporal poles. METHODS: A total of 23 patients (14 male and 9 female) with a mean age of 25.2 years (range 17-45 years) were included in this study, which was conducted over a 4-year period. All patients suffered medically refractory epilepsy due to unilateral, MRI-proven MTS, with no other imaging abnormalities. All participants underwent preoperative single-voxel proton MRS using a 3-T MRI unit. The hippocampi and temporal poles were examined bilaterally. The concentrations of N-acetyl-aspartate (NAA), choline (Cho), and creatine (Cr) were measured, and the NAA/Cho, NAA/Cr, and NAA/Cho+Cr ratios were calculated. All patients underwent anterior temporal lobectomy and ipsilateral amygdalohippocampectomy, and surgical specimens from the temporal poles were sent for histopathological examination. Comparisons of the spectroscopic and histopathological results of the resected temporal poles were performed. The modified Engel classification system was used for evaluating seizure outcome in the cohort. RESULTS: The preoperative spectroscopic profiles of the sclerotic hippocampi were abnormal in all patients, and the contralateral hippocampus showed altered spectroscopic findings in 12 patients (52.2%). Spectroscopy of the temporal poles demonstrated severely decreased concentrations of NAA, markedly increased concentrations of Cho, and increased concentrations of Cr in the temporal pole ipsilateral to the MTS in 15 patients (65.2%). Similarly, the NAA/Cho, NAA/Cr, and NAA/Cho+Cr ratios were severely decreased in the temporal pole ipsilateral to the MTS in 16 patients (69.6%). Histopathological examination of the resected temporal poles demonstrated ischemic changes in 5 patients (21.7%), gliotic changes in 4 (17.4%), demyelinating changes in 3 (13.0%), and microdysplastic changes in 1 patient (4.3%). Comparisons of the spectroscopic and histopathological findings showed that the sensitivity of proton MRS was 100%, its specificity was 80%, its positive predictive value was 87%, and its negative predictive value was 100%. The mean follow-up time in this study was 3.4 years. At the end of the 2nd postoperative year, 17 patients (73.9%) were in Engel Class I, 5 (21.7%) were in Class II, and 1 (4.3%) was in Class III. CONCLUSIONS: Proton MRS detected altered ipsilateral temporal pole metabolism in patients with unilateral MTS. These metabolic changes were associated with permanent histological abnormalities of the temporal pole. This finding demonstrates that MTS may be a more diffuse histological process, and exact preoperative knowledge of its temporal extent becomes of paramount importance in the selection of the best surgical approach in these patients. Further validation of the observations is necessary for defining the role of temporal pole proton MRS in cases of temporal lobe epilepsy.

A seminal fluid protease activates sperm motility in C. elegans males.

Seminal fluid factors have been shown to play a significant role in fertility in many animals. However, little is known about the contributions of seminal fluid to male fertility in C. elegans. In this commentary, we summarize our recent finding of a seminal fluid sperm activator, the serine protease TRY-5. TRY-5 is required for males to activate sperm, yet surprisingly it is not required for male fertility, likely due to redundancy with an activator present in hermaphrodites. TRY-5 is transferred to hermaphrodites during mating in a series of distinct release events just prior to transfer of sperm. Thus, we propose a model in which TRY-5 cleaves sperm cell surface proteins to trigger sperm maturation. We discuss other possible roles for seminal fluid factors in C. elegans and prospects for using TRY-5 as a marker for studies of male mating behavior and seminal fluid secretion.

TRY-5 is a sperm-activating protease in Caenorhabditis elegans seminal fluid.

Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction.

Scalp EEG does not predict hemispherectomy outcome.

BACKGROUND: Functional hemispherectomy is effective in carefully selected patients, resulting in a reduction of seizure burden up to complete resolution, improvement of intellectual development, and developmental benefit despite possible additional neurological deficit. Despite apparent hemispheric pathology on brain magnetic resonance imaging (MRI) or other imaging tests, scalp electroencephalography (EEG) could be suggestive of bilateral ictal onset or even ictal onset contralateral to the dominant imaging abnormality. We aimed to investigate the role of scalp EEG lateralization pre-operatively in predicting outcome. METHODS: We retrospectively reviewed 54 patients who underwent hemispherectomy between 1991 and 2009 at Medical College of Georgia (1991-2006) and Cincinnati Children's Hospital Medical Center (2006-2009) and had at least one year post-operative follow-up. All preoperative EEGs were reviewed, and classified as either lateralizing or nonlateralizing, for both ictal and interictal EEG recordings. RESULTS: Of 54 patients, 42 (78%) became seizure free. Twenty-four (44%) of 54 had a nonlateralizing ictal or interictal EEG. Further analysis was based on etiology of epilepsy, including malformation of cortical development (MCD), Rasmussen syndrome (RS), and stroke (CVA). EEG nonlateralization did not predict poor outcome in any of the etiology groups evaluated. CONCLUSION: Scalp EEG abnormalities in contralateral or bilateral hemispheres do not, in isolation, predict a poor outcome from hemispherectomy. Results of other non-invasive and invasive evaluations should be used to determine candidacy.

Closed-loop stimulation in the control of focal epilepsy of insular origin.

BACKGROUND: Previous studies have shown that closed-loop or responsive neurostimulation can abort induced or spontaneous epileptiform discharges. OBJECTIVE: To assess the effectiveness of a programmable cranially implanted closed-loop neurostimulation system in the control of seizures originating from an area relatively inaccessible by open craniotomy. METHOD: A patient with drug-resistant partial epilepsy had previously undergone open resection of the left frontal opercular cortex and the underlying insular area. Although subdural-depth electrode ictal recordings had been nonlocalizing, depth electrode insular stimulation had produced the patient's habitual aura. Postoperatively, there was a sustained 50% reduction in seizure frequency. The residual seizures were identical to the preoperative seizures. Repeat depth electrode monitoring revealed that the ictal focus was immediately posterior to the previously resected insular area. A closed-loop cranial internal pulse generator system including left anterior insular and posterior orbitofrontal depth electrodes was implanted. RESULT: There was an additional 60% reduction of seizures. CONCLUSION: Preliminary observation indicates that responsive neurostimulation may be an effective alternative to higher-risk resective epilepsy surgery.

Does registration of serial MRI improve diagnosis of dementia?

INTRODUCTION: We aimed to assess the value of a second MR scan in the radiological diagnosis of dementia. METHODS: One hundred twenty subjects with clinical follow-up of at least 1 year with two scans were selected from a cognitive disorders clinic. Scans were reviewed as a single first scan (method A), two unregistered scans presented side-by-side (method B) and a registered pair (method C). Scans were presented to two neuroradiologists and a clinician together with approximate scan interval (if applicable) and age. Raters decided on a main and subtype diagnosis. RESULTS: There was no evidence that differences between methods (expressed as relative odds of a correct response) differed between reviewers (p = 0.17 for degenerative condition or not, p = 0.5 for main diagnosis, p = 0.16 for subtype). Accordingly, results were pooled over reviewers. For distinguishing normal/non-progressors from degenerative conditions, the proportions correctly diagnosed were higher with methods B and C than with A (p = 0.001, both tests). The difference between method B and C was not statistically significant (p = 0.18). For main diagnosis, the proportion of correct diagnoses were highest with method C for all three reviewers; however, this was not statistically significant comparing with method A (p = 0.23) or with method B (p = 0.16). For subtype diagnosis, there was some evidence that method C was better than method A (p = 0.01) and B (p = 0.048). CONCLUSIONS: Serial MRI and registration may improve visual diagnosis in dementia.

Correlation between magnetic resonance imaging and histopathologic grades in Rasmussen syndrome.

The aim of this study was to investigate the correlation between magnetic resonance imaging (MRI) and histopathologic findings in Rasmussen syndrome. Serial MRIs were obtained for five patients who had histologically proven Rasmussen syndrome. The histopathologic grades of the lesions were subdivided into phases: active 1-3, resolving 1-3, and chronic inflammatory. The images were then correlated with histopathologic findings. Neuropathologic findings in the central areas on MRI demonstrated only the chronic and resolving grades, but active inflammatory abnormalities were present not only at the margins of the lesions, but also in areas of subtle signal abnormality on MRI. Atrophic areas on MRI exhibited all grades of histopathologic abnormalities, but chronic and resolving grades were predominant. Seizure duration of less than 6 months was associated with very active grades, duration of 1-2 years with variable grades, and duration greater than 6 years with chronic and resolving grades only. The MRI images correlated highly with histopathologic analysis. These findings suggest that the lesions initially arise from one site in the brain, and so support the centrifugal spreading theory of this disease. Findings also suggest that the margin rather than the center of the MRI abnormality may be the most ideal site for biopsy.

Historic evolution of open cingulectomy and stereotactic cingulotomy in the management of medically intractable psychiatric disorders, pain and drug addiction.

Stereotactic cingulotomy constitutes a psychosurgical procedure nowadays advocated in the treatment of medically intractable obsessive-compulsive disorder, chronic pain and drug addiction. From its theoretical conception to the first cingulectomies performed and modern stereotactic-guided cingulotomies, various target localization methods, different surgical techniques, and numerous lesioning devices have been utilized. In the current article, the authors performed a literature review related to cingular lesion placement in an effort to identify misconceptions of the past, recapitulate existing knowledge and recognize targets for further research. The initial animal and human electrophysiologic experimental data regarding the role of the cingulate cortex in various behavioral and cognitive functions were meticulously reviewed. The clinical indications, surgical technique and the clinical results and complications of open cingulectomies were examined. The anatomic target localization methodologies, surgical technique, and the outcome of the initial stereotactic cingulotomy procedures were reviewed, and the evolution of the imaging techniques, stereotactic devices, and lesioning strategies were followed. The modern advanced surgical techniques, clinical outcome and the procedure-associated complications were analyzed with particular emphasis on the emotional, behavioral, and cognitive procedure-induced changes. Large-scale prospective studies with strict inclusion and well-defined, objective outcome criteria are necessary for defining the role of stereotactic cingulotomy in the current psychosurgical armamentarium.

Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration.

Silencing and variegated transgene expression are poorly understood problems that can interfere with gene function studies in human embryonic stem cells (hESCs). We show that transgene expression (enhanced green fluorescent protein [EGFP]) from random integration sites in hESCs is affected by variegation and silencing, with only half of hESCs expressing the transgene, which is gradually lost after withdrawal of selection and differentiation. We tested the hypothesis that a transgene integrated into the adeno-associated virus type 2 (AAV2) target region on chromosome 19, known as the AAVS1 locus, would maintain transgene expression in hESCs. When we used AAV2 technology to target the AAVS1 locus, 4.16% of hESC clones achieved AAVS1-targeted integration. Targeted clones expressed Oct-4, stage-specific embryonic antigen-3 (SSEA3), and Tra-1-60 and differentiated into all three primary germ layers. EGFP expression from the AAVS1 locus showed significantly reduced variegated expression when in selection, with 90% +/- 4% of cells expressing EGFP compared with 57% +/- 32% for randomly integrated controls, and reduced tendency to undergo silencing, with 86% +/- 7% hESCs expressing EGFP 25 days after withdrawal of selection compared with 39% +/- 31% for randomly integrated clones. In addition, quantitative polymerase chain reaction analysis of hESCs also indicated significantly higher levels of EGFP mRNA in AAVS1-targeted clones as compared with randomly integrated clones. Transgene expression from the AAVS1 locus was shown to be stable during hESC differentiation, with more than 90% of cells expressing EGFP after 15 days of differentiation, as compared with approximately 30% for randomly integrated clones. These results demonstrate the utility of transgene integration at the AAVS1 locus in hESCs and its potential clinical application.

Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm.

Nodal, a member of the TGF-beta family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of beta-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of beta-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.