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Epigenetic modifications of chromatin play a critical role in regulating the fidelity of the genetic code and in controlling the translation of genetic information into the protein components of the cell. One key posttranslational modification is acetylation of histone lysine residues. Molecular dynamics simulations, and to a smaller extent experiment, have established that lysine acetylation increases the dynamics of histone tails. However, a systematic, atomic resolution experimental investigation of how this epigenetic mark, focusing on one histone at a time, influences the structural dynamics of the nucleosome beyond the tails, and how this translates into accessibility of protein factors such as ligases and nucleases, has yet to be performed. Herein, using NMR spectroscopy of nucleosome core particles (NCPs), we evaluate the effects of acetylation of each histone on tail and core dynamics. We show that for histones H2B, H3, and H4, the histone core particle dynamics are little changed, even though the tails have increased amplitude motions. In contrast, significant increases to H2A dynamics are observed upon acetylation of this histone, with the docking domain and L1 loop particularly affected, correlating with increased susceptibility of NCPs to nuclease digestion and more robust ligation of nicked DNA. Dynamic light scattering experiments establish that acetylation decreases inter-NCP interactions in a histone-dependent manner and facilitates the development of a thermodynamic model for NCP stacking. Our data show that different acetylation patterns result in nuanced changes to NCP dynamics, modulating interactions with other protein factors, and ultimately controlling biological output.

The HMQC pulse sequence and variants thereof have been exploited in studies of high molecular weight protein complexes, taking advantage of the fact that fast and slow relaxing magnetization components are sequestered along two distinct magnetization transfer pathways. Despite the simplicity of the HMQC scheme an even shorter version can be designed, based on elimination of the terminal refocusing period, as a further means of increasing signal. Here we present such an experiment, and show that significant sensitivity gains, in some cases by factors of two or more, are realized in studies of proteins varying in molecular masses from 100 kDa to 1 MDa.

The periplasmic protein DegP, that is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that maintains protein homeostasis in gram-negative bacteria. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo-oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming cage ensembles that can include extremely large cage particles. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of flexible cage structures including novel 30mer and 60mer particles. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size.Significance statementGram-negative pathogens export virulence factors that interfere with the function of host cells. This process is mediated by DegP, a protein which controls protein homeostasis in the periplasm of these bacteria and thus is a target for the development of novel antibiotics. DegP operates by incorporating client proteins inside cage-like structures to either recycle them or protect them from aggregation. Using a combination of dynamic light scattering measurements and cryogenic electron microscopy, we have shown that DegP can adopt many types of cages, some as large as subcellular organelles, depending on the size of the engaged client. This property likely enables DegP to capture different sized clients in response to protein misfolding stresses.

Weak macromolecular interactions assume a dominant role in the behavior of highly concentrated solutions, and are at the center of a variety of fields ranging from colloidal chemistry to cell biology, neurodegenerative diseases, and manufacturing of protein drugs. They are frequently measured in different biophysical techniques in the form of second virial coefficients, and nonideality coefficients of sedimentation and diffusion, which may be related mechanistically to macromolecular distance distributions in solution and interparticle potentials. A problem arises for proteins where reversible self-association often complicates the concentration-dependent behavior, such that grossly inconsistent coefficients are measured in experiments based on different techniques, confounding quantitative conclusions. Here we present a global multi-method analysis that synergistically bridges gaps in resolution and sensitivity of orthogonal techniques. We demonstrate the method with a panel of monoclonal antibodies exhibiting different degrees of self-association. We show how their concentration-dependent behavior, examined by static and dynamic light scattering and sedimentation velocity, can be jointly described in a self-consistent framework that separates nonideality coefficients from self-association properties, and thereby extends the quantitative interpretation of nonideality coefficients to probe dynamics in highly concentrated protein solutions.

The human high-temperature requirement A2 (HtrA2) protein is a trimeric protease that cleaves misfolded proteins to protect cells from stresses caused by toxic, proteinaceous aggregates, and the aberrant function of HtrA2 is closely related to the onset of neurodegenerative disorders. Our methyl-transverse relaxation optimized spectroscopy (TROSY)–based NMR studies using small-peptide ligands have previously revealed a stepwise activation mechanism involving multiple distinct conformational states. However, very little is known about how HtrA2 binds to protein substrates and if the distinct conformational states observed in previous peptide studies might be involved in the processing of protein clients. Herein, we use solution-based NMR spectroscopy to investigate the interaction between the N-terminal Src homology 3 domain from downstream of receptor kinase (drk) with an added C-terminal HtrA2-binding motif (drkN SH3-PDZbm) that exhibits marginal folding stability and serves as a mimic of a physiological protein substrate. We show that drkN SH3-PDZbm binds to HtrA2 via a two-pronged interaction, involving both its C-terminal PDZ-domain binding motif and a central hydrophobic region, with binding occurring preferentially via an unfolded ensemble of substrate molecules. Multivalent interactions between several clients and a single HtrA2 trimer significantly stimulate the catalytic activity of HtrA2, suggesting that binding avidity plays an important role in regulating substrate processing. Our results provide a thermodynamic, kinetic, and structural description of the interaction of HtrA2 with protein substrates and highlight the importance of a trimeric architecture for function as a stress-protective protease that mitigates aggregation.

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy-based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding-incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson's disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a “resting” hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP’s substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.

Solution NMR spectroscopy is widely used to investigate the thermodynamics and kinetics of the binding of ligands to their biological receptors, as it provides detailed, atomistic information, potentially leading to microscopic affinities for each binding event, and, to the development of allosteric pathways describing how the binding at one site affects distal sites in the molecule. Importantly, weak interactions that are often invisible to other biophysical methods can also be probed. Methodological advancements in NMR have enabled the investigation of high molecular weight, homo-oligomeric complexes that bind multiple ligand molecules, with increasing numbers of studies of the structural dynamics and binding properties of these systems. It therefore becomes of interest to consider how binding and kinetics parameters can be extracted from experiments on these more complicated molecules. Here we present the theoretical framework for analyzing binding reactions of homo-oligomeric complexes by NMR, taking into account all of the chemical species in solution and their corresponding NMR observables. A number of simulations are presented to illustrate the utility of the derived expressions.

G-quadruplexes (GQs) are four-stranded nucleic acid secondary structures formed by guanosine (G)-rich DNA and RNA sequences. It is becoming increasingly clear that cellular processes including gene expression and mRNA translation are regulated by GQs. GQ structures have been extensively characterized, however little attention to date has been paid to their conformational dynamics, despite the fact that many biological GQ sequences populate multiple structures of similar free energies, leading to an ensemble of exchanging conformations. The impact of these dynamics on biological function is currently not well understood. Recently, structural dynamics have been demonstrated to entropically stabilize GQ ensembles, potentially modulating gene expression. Transient, low-populated states in GQ ensembles may additionally regulate nucleic acid interactions and function. This review will underscore the interplay of GQ dynamics and biological function, focusing on several dynamic processes for biological GQs and the characterization of GQ dynamics by nuclear magnetic resonance (NMR) spectroscopy in conjunction with other biophysical techniques. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

The complex folding energy landscape of DNA G-quadruplexes leads to numerous conformations for this functionally important class of noncanonical DNA structures. A new layer of conformational heterogeneity comes from sequences with different numbers of G-nucleotides in each of the DNA G-strands that form the four-stranded G-quartet core. The mechanisms by which G-quadruplexes transition from one folded conformation to another are currently unknown. To address this question, we studied two different G-quadruplexes, selecting a single conformation by blocking hydrogen bonding with photolabile protection groups. Upon irradiation, the block can be released and the kinetics of re-equilibration to the native conformational equilibrium can be determined by time-resolved NMR. We compared the NMR-derived refolding kinetics with data derived from thermal hysteresis folding kinetic experiments and found excellent agreement. The outlined methodological approach allows separation of K+-induced G-quadruplex formation and subsequent refolding and provides key insight into rate-limiting steps of G-quadruplex conformational dynamics.

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2'-endo conformation, instead of the C3'-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology., Funding for open access charge: NSERC Discovery grant (to M.J.D., A.K.M.); CIHR DDTP Training Grant (to H.A., R.H.V.); MINECO [BFU2014-52864-R to C.G.]; CSIC-JAE contract (to N.M.P.).

G-quadruplexes (GQs) are 4-stranded DNA structures formed by tracts of stacked, Hoogsteen-hydrogen bonded guanosines. GQs are found in gene promoters and telomeres where they regulate gene transcription and telomere elongation. Though GQ structures are well-characterized, many aspects of their conformational dynamics are poorly understood. For example, when there are surplus guanosines in some of the tracts, they can slide with respect to one another, a process we term G-register (GR) exchange. These motions could in principle entropically stabilize the folded state, crucially benefitting GQs as their stabilities are closely tied to biological function. We have developed a method for characterizing GR exchange where each isomer in the wild-type conformational ensemble is trapped by mutation and thermal denaturation data for the set of trapped mutants and wild-type are analyzed simultaneously. This yields GR isomer populations as a function of temperature, quantifies conformational entropy and sheds light on correlated sliding motions of the G-tracts. We measured entropic stabilizations from GR exchange up to 14.3 ± 1.6 J mol(-1) K(-1), with melting temperature increases up to 7.3 ± 1.6°C. Furthermore, bioinformatic analysis suggests a majority of putative human GQ sequences are capable of GR exchange, pointing to the generality of this phenomenon.

Surgical techniques may vary extensively between centers. We report on a web-based survey aimed at evaluating the current technical approaches in different centers around the world performing epilepsy surgery in children. The intention of the survey was to establish technical standards. A request was made to 88 centers to complete a web-based survey comprising 51 questions. There were 14 questions related to general issues, 13 questions investigating the different technical aspects for children undergoing epilepsy surgery, and 24 questions investigating surgical strategies in pediatric epilepsy surgery. Fifty-two centers covering a wide geographic representation completed the questionnaire. The median number of resective procedures per center per year was 47. Some important technical practices appeared (>80% of the responses) such as the use of prophylactic antibiotics (98%), the use of high-speed drills for bone opening (88%), nonresorbable material for bone flap closure (85%), head fixation (90%), use of the surgical microscope (100%), and of free bone flaps. Other questions, such as the use of drains, electrocorticography (ECoG) and preoperative withdrawal of valproate, led to mixed, inconclusive results. Complications were noted in 3.8% of the patients submitted to cortical resection, 9.9% hemispheric surgery, 5% callosotomy, 1.8% depth electrode implantation, 5.9% subdural grids implantation, 11.9% hypothalamic hamartoma resection, 0.9% vagus nerve stimulation (VNS), and 0.5% deep brain stimulation. There were no major differences across regions or countries in any of the subitems above. The present data offer the first overview of the technical aspects of pediatric epilepsy surgery worldwide. Surprisingly, there seem to be more similarities than differences. That aside many of the evaluated issues should be examined by adequately designed multicenter randomized controlled trials (RCTs). Further knowledge on these technical issues might lead to increased standardization and lower costs in the future, as well as definitive practice guidelines.

Differential scanning calorimetry (DSC) is a powerful technique for measuring tight biomolecular interactions. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Using experimental data for two DNA aptamers that bind to the thermolabile ligand cocaine, we present a new global fitting analysis that yields the complete set of folding and binding parameters for the initial and final forms of the ligand from a pair of DSC experiments, while accounting for the thermal conversion. Furthermore, we show that the rate constant for thermolabile ligand conversion may be obtained with only one additional DSC dataset.

Glioneuronal tumours are an important cause of treatment-resistant epilepsy. Subtypes of tumour are often poorly discriminated by histological features and may be difficult to diagnose due to a lack of robust diagnostic tools. This is illustrated by marked variability in the reported frequencies across different epilepsy surgical series. To address this, we used DNA methylation arrays and RNA sequencing to assay the methylation and expression profiles within a large cohort of glioneuronal tumours. By adopting a class discovery approach, we were able to identify two distinct groups of glioneuronal tumour, which only partially corresponded to the existing histological classification. Furthermore, by additional molecular analyses, we were able to identify pathogenic mutations in BRAF and FGFR1, specific to each group, in a high proportion of cases. Finally, by interrogating our expression data, we were able to show that each molecular group possessed expression phenotypes suggesting different cellular differentiation: astrocytic in one group and oligodendroglial in the second. Informed by this, we were able to identify CCND1, CSPG4, and PDGFRA as immunohistochemical targets which could distinguish between molecular groups. Our data suggest that the current histological classification of glioneuronal tumours does not adequately represent their underlying biology. Instead, we show that there are two molecular groups within glioneuronal tumours. The first of these displays astrocytic differentiation and is driven by BRAF mutations, while the second displays oligodendroglial differentiation and is driven by FGFR1 mutations. Electronic supplementary material The online version of this article (doi:10.1007/s00401-017-1773-z) contains supplementary material, which is available to authorized users.

Aim To establish the rates and types of psychiatric disorder in children before and after surgery for extratemporal epilepsy. Relationships between psychiatric morbidity and demographic/clinical variables were examined. Method A retrospective case note review of 71 children undergoing extratemporal focal resection for drug resistant epilepsy in a specialist epilepsy surgery programme between 1997 and 2008. Psychiatric diagnoses were derived from pre- and postoperative assessments according to DSM-IV criteria. Results Seventy-one children (38 males, 33 females) were eligible for this study. Mean age (SD) at surgery was 9 (5) years. Frontal resections were performed in 73% of the children, parietal in 17%, and occipital in 10%. Mental health problems were present in 37 of 71 (52%) children pre- and/or postoperatively. A similar proportion of children had psychiatric diagnoses pre- and postoperatively: 31 of 71 (44%) and 32 of 71 (45%) respectively. Interpretation Psychopathology is common in children with extratemporal epilepsy. In this sample, the impact of surgery on psychiatric symptoms was not predictable: some children were unchanged, others improved, and others acquired new psychiatric diagnoses postoperatively. Given the high rates of psychiatric disorder in this group of patients, detection and treatment of mental health needs may be important.

Objective: Temporal lobe resection is an established treatment for medication-resistant temporal lobe epilepsy, which in recent years has increasingly been performed in children. However, little is known about the long-term outcome in these children. The aim of this study was to characterize intellectual and psychosocial functioning of children after temporal lobe resection as they progress into late adolescence and adulthood. Methods: We report the long-term follow-up of 42 children who underwent temporal lobe surgery after an average postoperative period of 9 years. Longitudinal change in IQ was documented, psychosocial outcome including quality of life was assessed, and preoperative and postoperative T1-weighted MRI brain scans were evaluated quantitatively. A well-matched nonsurgical comparison group of 11 children with similar clinical characteristics was also assessed. Results: At follow-up, 86% of the surgical group were seizure-free, and 57% were no longer taking antiepileptic medication. A significant increase in IQ was found in the surgical group after an extended follow-up period of >5 years. This IQ change was not found in the nonsurgical comparison group. IQ increases were associated with cessation of antiepileptic medication and changes in MRI-derived gray matter volume. The surgical group also reported better psychosocial outcome including quality of life, which was more strongly associated with seizure freedom rather than surgery per se. Conclusions: Surgery for temporal lobe epilepsy performed in childhood results in excellent long-term seizure control and favorable cognitive outcome along with positive effects on brain development. Classification of evidence: This study provides Class III evidence that temporal lobectomy in children with temporal lobe epilepsy is associated with improved long-term intellectual outcomes compared with those undergoing standard medical treatment.

The heterogeneity of tumours and uncertainties surrounding derived short-term cell cultures and established cell lines fundamentally challenge the research and understanding of tumour growth and development. When tumour cells are cultured, changes are inevitably induced due to the artificial growth conditions. Several recent studies have questioned how representative established cell lines or derived short-term cell cultures are of the tumour in situ. We have characterised gene expression changes induced by short-term culture in astrocytoma in order to determine whether derived short-term cell cultures are representative of the tumour in situ. In comparison to the majority of studies, paired biopsies and derived short-term cultures were investigated to reduce the effects of long-term culture and inter-tumour variability when comparing biopsies and derived cultures from tumours with the same histology from different individuals. We have used the Affymetrix GeneChip® U133A to generate gene expression profiles of 6 paediatric pilocytic astrocytoma (PA) biopsies and derived short-term cell cultures and 3 adult glioblastoma multiforme (GBM) biopsies and derived short-term cultures. Significant differential gene expression is induced by short-term culture. However, when the biopsy and derived short-term cell culture samples were grouped according to tumour type (PA and GBM) a molecular signature of 608 genes showed significant differential expression between the groups. This gene cohort can distinguish PA and GBM tumours, regardless of the sample source, suggesting that astrocytoma derived short-term cultures do retain key aspects of the global tumour expression profile and are representative of the tumour in situ. Furthermore, these genes are involved in pathways and functions characteristic of adult GBM including VEGF signalling, hypoxia and TP53 signalling.

SPECT can be used to image regional cerebral blood flow (rCBF) and has been shown to help localize the seizure focus in partial epilepsies as part of the presurgical evaluation. Few studies have explored the possible relation between preoperative SPECT and underlying pathology, or any relation to postsurgical outcome. In this study preoperative ictal and interictal rCBF in relation to the histopathological diagnosis and outcome in a series of 35 children (24 females, 11 males; mean age 9.6 years, age range 11 months to 18 years) who had undergone resective surgery for epilepsy were retrospectively evaluated. A correlation between ictal hyperperfusion and the underlying responsible pathology was shown, with a consistent ictal increase in perfusion in developmental pathologies and Rasmussen's encephalitis, and consistent interictal hypoperfusion in hippocampal sclerosis (HS). No rCBF study parameter appeared to relate to outcome but in the group with HS the best outcome was seen in those with localizing ictal rCBF. The varied group of pathologies from hemispherectomy had excellent outcome but the SPECT findings had little to contribute over the abnormalities detected on MRI. In conclusion, rCBF studies remain a useful presurgical investigation in children with partial epilepsy, especially where HS, cortical dysplasia, or inflammatory disease are the underlying pathology. However, rCBF studies add little to the investigation of children with seizures secondary to benign tumours or cerebral infarcts, or where hemispherectomy is the likely preferred surgical option.

Balloon cells (BC) are the prominent and defining cellular component of type IIB Focal Cortical Dysplasia (FCD), a common cause of focal epilepsy in patients undergoing surgical treatment. BC are considered immature cells of uncommitted cellular differentiation having immunophenotypical characteristics of both neurones and glia. They are often located in the lower cortical layers and white matter underlying the dysplastic cortex, suggesting migratory arrest during development. We investigated the proliferative potential of BC in 15 cases of FCD from patients with a wide range of ages using immunohistochemistry for Mcm2 (mini chromosome maintenance protein) and Ki67. In the majority of cases, BC showed Mcm2 nuclear positivity. In addition, cells with intermediate neuronal-glial characteristics were labelled whilst the dysmorphic or hypertrophic pyramidal neuronal components of FCD were not. Ki67 labelled only occasional BC. These findings support the view that BC cells represent a pool of less differentiated glial cells with proliferative capacity which may have potential for delayed neuronal differentiation. Furthermore, as Mcm2 specifically identifies BC populations, this marker may be of diagnostic value in the subtyping of FCD lesions in patients with epilepsy.

Surgical approaches to the posterior fossa may be complicated by pseudomeningocoele formation. We report on its natural history and risk factors for its formation, as seen on serial MRI postoperatively in children with posterior fossa tumours. In a retrospective study of 84 children undergoing surgery for posterior fossa tumours, 13 (16%) developed clinically apparent pseudomeningocoeles. On postoperative MRI, pseudomeningocoeles were apparent in 34 (41%) patients at 1-5 days, but in only four patients at 10-15 months postsurgery. There was a progressive decrease in the mean depth of pseudomeningocoele measured from the MRI scans postoperatively. Patients with pseudomeningocoeles were more likely to have a postoperative CSF leak from the wound (39 v. 13%), lumbar punctures or lumbar drains (54 v. 25%), wound re-closures (23 v. 1%) and prolonged hospital stay (19.9 v. 14.5 days). On multivariate analysis, patients with pseudomeningocoeles were also more likely to have undergone a suboccipital craniectomy than those without pseudomeningocoeles (69 v. 38%). Postoperative pseudomeningocoele formation following posterior fossa surgery is more apparent radiologically than clinically, but there is clinical and radiological evidence that pseudomeningocoeles gradually resolve over the postoperative period. The risk of pseudomeningocoele formation is increased by performing a suboccipital craniectomy and there is an association with increased CSF leaks, needing re-closure of the wounds.

Increasingly, Onyx is used for endovascular embolization of aneurysms and arterio-venous malformations. Although reports in the literature on the use of Onyx are favourable, there have been so far no reports on the central nervous system (CNS) infection rate after embolisation with Onyx and no recommendations as to the management of these infections. We present two cases of paediatric patients who acquired CNS infection with Pseudomonas aeruginosa after Onyx embolisation of AVMs and describe their subsequent management. Presence of established infection after Onyx embolisation should be dealt with by removal of infected material, administration of appropriate antibiotic therapy and supportive treatment.

Background: Epilepsy carries an increased risk of premature death. For some people with intractable focal epilepsy, surgery offers hope for a seizure-free life. The authors aimed to see whether epilepsy surgery influenced mortality in people with intractable epilepsy. Methods: The authors audited survival status in two cohorts (those who had surgery and those who had presurgical assessment but did not have surgery). Results: There were 40 known deaths in the non-surgical group (3365 person years of follow-up) and 19 in the surgical group (3905 person-years of follow-up). Non-operated patients were 2.4 times (95% CI 1.4 to 4.2) as likely to die as those who had surgery. They were 4.5 times (95% CI 1.9 to 10.9) as likely to die a probable epilepsy-related death. In the surgical group, those with ongoing seizures 1 year after surgery were 4.0 (95% CI 1.2 to 13.7) times as likely to die as those who were seizure-free or who had only simple partial seizures. Time-dependent Cox analysis showed that the yearly outcome group did not significantly affect mortality (HR 1.3, 95% CI 0.9 to 1.8). Conclusion: Successful epilepsy surgery was associated with a reduced risk of premature mortality, compared with those with refractory focal epilepsy who did not have surgical treatment. To some extent, the reduced mortality is likely to be conferred by inducing freedom from seizures. It is not certain whether better survival is attributable only to surgery, as treatment decisions were not randomised, and there may be inherent differences between the groups.

The aim of this study was to examine the range of sensitivity of a panel of short-term cultures derived from different types of malignant childhood brain tumours including medulloblastoma, ependymoma and glioblastoma multiforme to three cytotoxic drugs, lomustine (CCNU), vincristine (VCR) and procarbazine (PCB). Sensitivity was assessed using a modification of the dimethylthiazolyl-2,5-diphenyl tetrazolium bromide (MTT) assay. Short-term cell lines derived from ependymomas were considerably more resistant to VCR than other types of childhood brain tumours, while cultures derived from supratentorial primitive neuroectodermal tumour (PNET) displayed marked sensitivity to both lomustine and VCR. Cultures from ependymomas, medulloblastoma and astrocytic gliomas had similar sensitivity to lomustine and PCB as cultures derived from adult malignant astrocytoma.

Epileptic nystagmus (EN) is a rare form of nystagmus that occurs only during epileptic seizures. We report an infantile case in which EN was first noted at 10 days of age. Electronystagmography showed a right-beating nystagmus with predominantly linear slow phases that traversed the midline. Neuro-imaging revealed dysplasia of the left middle temporal gyrus extending posteriorly into the parieto-occipital cortex. The right hemisphere and subcortical structures appeared normal. Perfusion studies demonstrated interictal hypoperfusion with ictal hyperperfusion in the left temporal lobe. Electrocorticography demonstrated spiking over the left temporal-parieto-occipital region. Following extensive surgical resection of this area and weaning of anti-convulsants, the child has remained seizure-free without nystagmus. This case demonstrates the cortical origin of EN, and shows that infant cortex has functioning efferent connections to brainstem oculomotor centres from 10 days of age.

Study Design. All cervical spine radiographs of 44 patients with Crouzon syndrome treated at Great Ormond Street Hospital during the past 10 years were studied. Objectives. To assess the incidence and pattern of cervical spine abnormalities of patients with a confirmed diagnosis of Crouzon syndrome, but particularly regarding progressive fusion. Summary of Background Data. Previous studies into the cervical spine anomalies in those with Crouzon syndrome have shown an increased incidence of congenital abnormalities compared with the normal population. There is some suggestion from previous studies that cervical spine fusions are progressive in nature. Methods. All radiographs were reviewed by the craniofacial team, along with a single pediatric radiologist with experience in assessment of skeletal dysplasias. Results. Radiologic abnormalities included "butterfly" vertebrae and fusions of the bodies and the posterior elements. Evidence of fusion was present in eight of 44 (18%) of patients. C2-C3 and C5-C6 were almost equally affected. Block fusions involving multiple vertebrae were seen. Analysis of sequential studies in 16 patients showed evidence of progression in five. Conclusions. These results reveal an incidence of fusions that is lower than reported previously. There is radiologic evidence from serial studies that the fusions are progressive, and because these patients are children, the fusion process may not be complete, which may account for the lower incidence of fusions than in previous studies. The pattern of fusions is different from that in earlier studies, which may be a result of the method of diagnosis because this population is less likely to include atypical forms of other syndromes (which have a higher incidence of cervical fusions, particularly at C2-C3). Regarding other congenital anomalies, it appears that butterfly vertebrae are especially prevalent in association with Crouzon syndrome.

Twenty patients with a diagnosis of Saethre-Chotzen syndrome had their cervical spine radiographs reviewed. Radiologic abnormalities including vertebral fusion were present in 9 of the 20 patients. Fusion of both the vertebra! bodies and the posterior elements was noted, although the latter site was more common. C2–3 was the level most commonly involved, although other levels were recorded. Analysis of sequential radiographs in nine patients revealed evidence of progression In seven patients. In those studies in children aged under 2 years, only 1 of 18 films showed evidence of fusion, while in those over 2 years of age, 10 of 12 showed evidence of fusion. These results reveal that the incidence of cervical anomalies in Saethre-Chotzen syndrome is greater than that in the general population. There is both direct and indirect evidence that the vertebral fusions are progressive during childhood.

Promoter hypermethylation and transcriptional silencing is a common epigenetic mechanism of gene inactivation in cancer. To identify targets of epigenetic silencing in paediatric intracranial ependymoma, we used a pharmacological unmasking approach through treatment of 3 ependymoma short-term cell cultures with the demethylating agent 5-Aza-2'-deoxycytidine followed by global expression microarray analysis. We identified 55 candidate epigenetically silenced genes, which are involved in the regulation of apoptosis, Wnt signalling, p53 and cell differentiation. The methylation status of 26 of these genes was further determined by combined bisulfite restriction analysis (COBRA) and genomic sequencing in a cohort of 40 ependymoma samples. The most frequently methylated genes were BEX1 (27/40 cases), BAI2 (20/40), CCND2 (18/40), and CDKN2A (14/40). A high correlation between promoter hypermethylation and decreased gene expression levels was established by real-time quantitative PCR, suggesting the involvement of these genes in ependymoma tumourigenesis. Furthermore, ectopic expression of brain-expressed X-linked 1 (BEX1) in paediatric ependymoma short-term cell cultures significantly suppressed cell proliferation and colony formation. These data suggest that promoter hypermethylation contributes to silencing of target genes in paediatric intracranial ependymoma. Epigenetic inactivation of BEX1 supports its role as a candidate tumour suppressor gene in intracranial ependymoma, and a potential target for novel therapies for ependymoma in children.

Propofol is used widely during general anaesthesia but there has been concern that it may be implicated in provoking seizure activity. We have investigated the effects of low-dose propofol on the electrocorticogram of anaesthetized patients undergoing surgery for medically intractable epilepsy. During continuous peroperative recording of the electrocorticogram, propofol was administered in 25 mg increments until burst suppression occurred. Activation of the electrocorticogram occurred in 17 of 20 patients. There was an increase in mean spike frequency in 16, extension of spike distribution in 15 and polyphasia in 13 patients. The mean dose of propofol required to cause burst suppression was 88.2 (range 25-175) mg. We conclude that at low doses, propofol caused activation of the electrocorticogram in epileptic patients but at higher doses burst suppression was induced.

Butylated hydroxytoluene is an antioxidant that has antiviral properties and sustains sperm viability during freezing and thawing. A field trial involving 11 bulls and 19,000 AI was conducted to determine whether addition of .5 mM butylated hydroxytoluene to whole milk extender during seminal processing affected bull fertility as estimated by nonreturn rates generated by cows bred to the bulls. Effects of bull, batch of semen nested within bull, treatment, and month of AI were studied. Nonreturn rates were recorded for each month for every bull, batch of semen (ejaculates pooled on a given day), and treatment combination. Because some bulls had6 batches of semen, the original experimental design was reduced to two smaller designs. Categorical data analysis with maximum likelihood estimation was used for analysis of nonreturn rates. The results from three models were used to interpret the data. The nonreturn rates were approximately 73.9% for the butylated hydroxytoluene treatment and 74.1% for the control. In all models, bull effect was significant, but batch, month of AI, and treatment had no effect on bull fertility. Addition of .5 mM butylated hydroxytoluene to whole milk extender during semen processing did not affect bull nonreturn rates.

Aspirated intracranial fluid, in the surgical management of intracranial sepsis, may not culture an organism due to the previous administration of antibiotics. We have sought to utilise polymerase chain reaction (PCR) to determine the cause of culture-negative sepsis and in monitoring response to therapy. This was a retrospective review of five cases of Streptococcus pneumoniae intracranial sepsis. Samples were analysed using real-time quantitative PCR targeting the pneumococcal lytA gene and the number of genome copies per microlitre of sample determined. Streptococcus pneumoniae sepsis was diagnosed by PCR in five culture-negative cases comprising: ventriculitis (×3), subdural empyema and meningitis. Serial serum inflammatory markers (CRP and WBC) and number of genome copies were graphically plotted over the duration of inpatient stay for cases requiring surgical drainage of recurrent collections or external ventricular drainage. A correlation was demonstrated between change in bacterial genomic load and serum inflammatory markers, reflecting similar changes in clinical state. This is the first report of the use of serial quantitative PCR in monitoring the course of intracranial sepsis secondary to S. pneumoniae. Further work is required to determine the precise relationship between serum inflammatory markers, clinical state and bacterial load: do changes in one precede the other? Furthermore, a threshold value for number of genome copies in cerebrospinal fluid/aspirate samples has yet to be defined.

The Westinghouse Small Modular Reactor (SMR) incorporates an integral pressurized water reactor (iPWR) design in which all components associated with the nuclear steam supply system are housed within one pressure vessel. The Westinghouse SMR design also utilizes many of the key features from the AP1000 ® plant, including passive safety systems. The Westinghouse SMR will be fueled by a derivative of the successful 17×17 Robust Fuel Assembly (RFA) product. An 89 assembly core with an active height of 8 feet will provide a 24 month operating cycle with a power output of 800 MWt. Derived from the AP1000 plant and adapted to operate inside the reactor pressure vessel, 37 control rod drive mechanisms provide reactor shutdown and reactivity control capabilities. Eight seal less pumps provide a nominal reactor coolant flow of 100,000 gallons per minute. An innovative evolution of a straight tube steam generator produces a saturated mixture that is delivered to a steam separating drum located outside of the containment vessel. The steam generator along with the integral pressurizer is attached to the reactor vessel with a single closure flange located near the center of gravity of the reactor assembly and is designed to be removed during refueling operations. Like the AP1000 plant, the Westinghouse SMR relies on the natural forces of gravity and natural circulation to provide core and containment cooling during accident conditions. The passive cooling systems provide sufficient heat removal for seven days without the need for offsite AC power sources. The Westinghouse SMR also includes traditional active components such as diesel generators and pumps; however these components are not required for the safe shutdown of the plant. At a diameter of 32 feet, approximately 25 of the Westinghouse SMR containment vessels can fit within the envelope of the AP1000 containment building. This compact containment will be completely submerged in water during power operation providing a heat sink for postulated accidents. For protection against external threats, the containment vessel and plant safety systems are located below ground level. At approximately one fifth the net electrical output of the AP1000 plant, the Westinghouse SMR is designed to address infrastructure challenges associated with replacing America’s aging fossil fuel plants by providing a safe, clean and reliable energy source. The challenges associated with economies of scale are offset with a compact and simplified plant design, rail shippable components and modular construction.Copyright © 2011 by ASME

Dysarthria following surgical resection of childhood posterior fossa tumour (PFT) is most commonly documented in a select group of participants with mutism in the acute recovery phase, thus limiting knowledge of post-operative prognosis for this population of children as a whole. Here we report on the speech characteristics of 13 cases seen long-term after surgical treatment for childhood PFT, unselected for the presence of post-operative mutism (mean time post-surgery=6y10m, range 1;4-12;6 years, two had post-operative mutism), and examine factors affecting outcome. Twenty-six age- and sex- matched healthy controls were recruited for comparison. Participants in both groups had speech assessments using detailed perceptual and acoustic methods. Over two-thirds of the group (69%) with removal of PFT had a profile of typically mild dysarthria. Prominent speech deficits included consonant imprecision, reduced rate, monopitch and monoloudness. We conclude that speech deficits may persist even up to 10 years post-surgery in participants who have not shown mutism in the acute phase. Of cases with unilateral lesions, poorer outcomes were associated with right cerebellar tumours compared to left, consistent with the notion based on adult data that speech is controlled by reciprocal right cerebellar/left frontal interactions. These results confirm the important role of the cerebellum in the control of fine speech movements in children.

A 30-year-old female presented with headache, CSF rhinorrhoea, mild right facial weakness, 2 months following temporal lobectomy for epilepsy. CT revealed marked intraventricular pneumocephalus with breached air cells in the pneumatized lower part of temporal bone. The dural and bony defects repaired successfully with complete resolution of the pneumocephalus.

Purpose Resective epilepsy surgery in early childhood has become an important treatment option for selected infants and children with epilepsy. We describe experience and clinical outcomes of children under 3 years undergoing epilepsy surgery at Great Ormond Street Hospital (GOSH). Methods All children under 36 months of age who had resective surgery for the purpose of treating epilepsy within the GOSH epilepsy surgery programme were ascertained using a departmental database. Aetiology, post-operative seizure frequency, pre and post-operative cognitive function, long-term complications and re-operation rates were analysed by retrospective examination of clinical records. Results Forty-two children were included in our cohort with a median age at surgery of 20 months (range 3–36 months). Surgical procedures comprised 25 functional hemispherectomies, two anatomical hemispherectomies, four multilobar resections, seven lobar resections and four focal resections. 7/42 (17%, 95% CI 8–31%) children underwent re-operation. 20/42 (48%, 95% CI 33–62%) children achieved seizure freedom. 18/42 (43%, 95% CI 29–58) demonstrated an improvement in seizure frequency and no children had an increase in seizure frequency. Post-operative complications included subsequent shunt procedure in 5/25 (20%, 95% CI 9–39%) children undergoing hemispherectomy. There were no mortalities. In 23 children pre- and post-operative DQ or IQ was determinable allowing longitudinal comparison. Five children had a decrease in DQ/IQ >15 and two children had an increase DQ/IQ >15. Discussion Epilepsy surgery in children under 3 years of age offers suitable candidates a good chance of significantly improved seizure outcome which compares with rates in older cohorts.

PURPOSE: Hippocampal sclerosis (HS) is the most common lesion underlying drug-resistant temporal lobe epilepsy. Whether HS is a developmental or acquired pathology remains unclear. Whereas HS has been causally linked to prolonged febrile convulsions in childhood, evidence also exists that it may coexist with extrahippocampal abnormalities, the concept of "dual pathology." The aims of this study were to address whether hippocampal abnormality consistent with HS (a) occurs in children with lesional extrahippocampal epilepsy, (b) is more commonly seen in association with developmental rather than acquired extrahippocampal pathologies, and (c) whether any effect of age at seizure onset is found on the occurrence of HS in lesional extrahippocampal epilepsy. METHODS: Clinical and histopathologic data of patients having resective surgery for extrahippocampal epilepsy that included the hippocampus were investigated. RESULTS: Twenty-nine children were retrospectively included in this study, and 21 (72%) of 29 were found to have a hippocampal abnormality consistent with HS. No relation was noted between developmental or acquired extrahippocampal pathologies and the presence of hippocampal abnormality. Children with normal hippocampi on visual histologic assessment had a significantly younger age at seizure onset (p < 0.001). Duration of epilepsy was not correlated with the presence of hippocampal abnormality. CONCLUSIONS: Hippocampal abnormalities are seen in similar proportions with both acquired and developmental extra-hippocampal pathologies, suggesting that these abnormalities are the result of seizures from the focus that is remote from the hippocampus. In addition, children who have their initial seizure at an early age are less likely to develop seizure-induced hippocampal injury.

The aim of this study was to establish the rate and spectrum of psychiatric disorder among children before and after temporal lobe surgery for epilepsy. Data were examined for associations between psychopathology and seizure outcome following surgery, or association between psychopathology and other variables, such as laterality of lesion, sex, cognitive level, and underlying pathology. Participants were 60 children (35 males, 25 females) who had focal seizures of temporal lobe origin and who had undergone temporal lobe resection between 1992 and 1998; mean age at time of operation 10 y 7 mo, (SD 4 y 11 mo) range 7 mo to 17 y 11 mo. Mean length of follow-up was 5.1 years (SD 2.3, range 2 to 10 y). Twenty-eight (47%) children had undergone right temporal lobectomy. Diagnosis of a psychiatric disorder was present in 50/60 (83%) children at some point, with high rates of psychiatric comorbidity. Common childhood psychiatric disorders of attention-deficit-hyperactivity disorder, oppositional defiant disorder/conduct disorder, and emotional disorders were present in about 25% of children. Disorders rarely seen in the general child population were over-represented: disruptive behaviour disorder--not otherwise specified (30/60 [50%]), and pervasive developmental disorder (autistic spectrum disorder; 23/60 [38%]). there was no significant relationship between pathology, sex, seizure frequency, or postoperative seizure outcome and psychiatric disorder, other than for pervasive developmental disorder. The same proportion of children had psychiatric diagnoses pre- and postoperatively (43/60 [72%] and 41/57 [72%] respectively). Although mental health problems are common in children undergoing temporal lobe resection, there are few predictors of psychiatric outcome following epilepsy surgery. Parents require counselling on these issues in the preoperative work-up.

Hemispherectomy has been performed in the treatment of epilepsy in association with hemiplegia for over 50 years. However, the optimal timing of surgery with respect to age at presentation and the influence of underlying pathology on outcome is only slowly emerging. This study reports on the clinical course and outcomes of 33 children who underwent hemispherectomy at Great Ormond Street Hospital, London, between 1991 and 1997. Age at surgery was 0.33-17 years (median 4.25) with 1-8 years follow-up (median 3.4). The underlying pathology was developmental in 16 (10 hemimegalencephaly, two polymicrogyria, two focal cortical dysplasia, one diffuse cortical dysplasia and one microdysgenesis), acquired in 11 (six middle cerebral artery infarct, three post encephalitis/trauma, and one each of hemiconvulsion-hemiplegia epilepsy and perinatal ischaemic insult) and progressive in six children (four Rasmussen encephalitis, two Sturge-Weber syndrome). At follow-up, 52% were seizure free, 9% experienced rare seizures, 30% showed75% reduction in seizures and 9% showed75% seizure reduction or no improvement. Seizure freedom was highest in those with acquired pathology (82%), followed by those with progressive pathology (50%) and those with developmental pathology (31%). However, seizure freedom, rare seizures or75% reduction in seizures occurred in 100% of those with progressive pathology, 91% of those with acquired and 88% of those with developmental pathology, indicating a worthwhile seizure outcome in all groups. Hemiplegia remained unchanged following surgery in 22 out of 33 children, improved in five and was worse in six. No significant cognitive deterioration or loss of language occurred, and four children showed significant cognitive improvement. Behavioural improvement was reported in 92% of those who had behaviour problems pre-operatively.

To evaluate the predictive value of symptoms, signs, and radiographic findings accompanying presumed ventriculoperitoneal (VP) shunt malfunction, by comparing presentation with operative findings and subsequent clinical course.Prospective study of all 53 patient referrals to a paediatric neurosurgical centre between April and November 1999 with a diagnosis of presumed shunt malfunction. Referral pattern, presenting symptoms and signs, results of computed tomography (CT) scanning, operative findings, and clinical outcome were recorded. Two patient groups were defined, one with proven shunt block, the other with presumed normal shunt function. Symptomatology, CT scan findings, and the subsequent clinical course for each group were then compared.Common presenting features were headache, drowsiness, and vomiting. CT scans were performed in all patients. Thirty seven had operatively proven shunt malfunction, of whom 34 had shunt block and three shunt infection; 84% with shunt block had increased ventricle size when compared with previous imaging. For the two patient groups (with and without shunt block), odds ratios with 95% confidence intervals on their presenting symptoms were headache 1.5 (0.27 to 10.9), vomiting 0.9 (0.25 to 3.65), drowsiness 10 (0.69 to 10.7), and fever 0.19 (0.03 to 6.95). Every patient with ventricular enlargement greater than their known baseline had a proven blocked shunt.Drowsiness is by far the best clinical predictor of VP shunt block. Headache and vomiting were less predictive of acute shunt block in this study. Wherever possible CT scan findings should be interpreted in the context of previous imaging. We would caution that not all cases of proven shunt blockage present with an increase in ventricle size.

Medulloblastoma is an infratentorial primitive neuroectodermal tumour. It is the most commonly occurring brain tumour of childhood, accounting for 15-20% of all paediatric tumours. Extracranial metastasis is rare, but may involve the skeleton. Jaw lesions, however, have never been described. A case is reported of metastases of a medulloblastoma to the jaw including the dental pulp.