Management of Status Epilepticus by Different Pediatric Departments: Neurology, Intensive Care, and Emergency Medicine.

INTRODUCTION: The aim of this study was to explore the differences in status epilepticus (SE) management among pediatric neurology, emergency medicine, and intensive care specialists in Turkey. METHODS: A 22-item questionnaire regarding first-, second-, and third-line management strategies of SE including demographic characteristics and common etiologies according to the specialty of participants was mailed to 370 specialists working in Turkey. RESULTS: A total of 334 participants (response rate 90%) comprising 136 pediatric neurologists, 102 pediatric emergency medicine specialists, and 96 pediatric intensive care specialists completed the survey. While intensive care specialists frequently managed SE due to metabolic and autoimmune reasons, the most common etiologies encountered by emergency medicine specialists were epilepsy and infections. More than half of the intensive care specialists (64.6%) reported using non-BZD antiseizure medications in the 5th minute of the seizure. Most of the neurologists (76.4%) preferred to administer intravenous (IV) levetiracetam infusion as a second-line agent. About half of intensive care specialists and neurologists tried immunomodulatory therapies in super-refractory SE. Intensive care and emergency medicine specialists were less likely to favor ketogenic diet and pyridoxine therapy for the treatment of super-refractory SE. The rate of requesting EEG monitoring to recognize nonconvulsive SE (NCSE) was found to be very low except for neurologists. CONCLUSION: There was no consensus among neurologists, intensive care specialists, and emergency medicine specialists in the management of SE in Turkey. Familiarity with particular antiseizure medications and the etiologies they manage seem to be the most important factors influencing the attitudes.

Corpus callosum thickness: A predictive factor for the first drug efficiency of self-limited epilepsy with centrotemporal spikes (selects)?

OBJECTIVE: To investigate the existence of a possible linkage between the thickness of corpus callosum (CC) regions and the first antiepileptic drug response in patients with Selects. MATERIALS AND METHODS: CC thickness of 68 patients with Selects and 42 healthy controls between 4 and 12 years of age were measured using brain magnetic resonance imaging (MRI). Clinical and EEG features of newly diagnosed Selects patients were recorded. Patients were divided into two groups: good-response (patients without seizures within 24 weeks) and poor-response (patients with ≥ 1 seizure within 24 weeks). Thickness of CC was compared between patients (good-response and poor-response groups).and healthy controls. RESULTS: The thicknesses of genu and isthmus were significantly reduced in the Selects group than healthy controls. Isthmus and splenium were significantly thinner in poor responders than those in the good-response group (p = 0.005 and p < 0.001, respectively). The total number of seizures was negatively correlated with the thickness of the body, isthmus, and splenium (p < 0.001). There was no significant difference in CC thickness of the children with and without electrical status epilepticus in sleep (ESES). The thickness of the isthmus and splenium were significantly thinner in patients receiving ≥ 2 antiepileptic drugs (p = 0.002 and p = 0.001, respectively). CONCLUSIONS: Our study highlights the notable differences in areas of CC in Selects patients. These changes may help uncover the underlying cause of seizure recurrence and antiepileptic drug (AED) response. Different thinner parts of CC may be a protective mechanism to prevent seizure spread to other brain regions. CC thickness can be used as a new radiologic biomarker for predicting first AED response and seizure recurrence in Selects patients.

Sub-millisecond chain collapse of the Escherichia coli globin ApoHmpH.

Myoglobins are ubiquitous proteins that play a seminal role in oxygen storage, transport, and NO metabolism. The folding mechanism of apomyoglobins from different species has been studied to a fair extent over the last two decades. However, integrated investigations of the entire process, including both the early (sub-ms) and late (ms-s) folding stages, have been missing. Here, we study the folding kinetics of the single-Trp Escherichia coli globin apoHmpH via a combination of continuous-flow microfluidic and stopped-flow approaches. A rich series of molecular events emerges, spanning a very wide temporal range covering more than 7 orders of magnitude, from sub-microseconds to tens of seconds. Variations in fluorescence intensity and spectral shifts reveal that the protein region around Trp120 undergoes a fast collapse within the 8 µs mixing time and gradually reaches a native-like conformation with a half-life of 144 µs from refolding initiation. There are no further fluorescence changes beyond ca. 800 µs, and folding proceeds much more slowly, up to 20 s, with acquisition of the missing helicity (ca. 30%), long after consolidation of core compaction. The picture that emerges is a gradual acquisition of native structure on a free-energy landscape with few large barriers. Interestingly, the single tryptophan, which lies within the main folding core of globins, senses some local structural consolidation events after establishment of native-like core polarity (i.e., likely after core dedydration). In all, this work highlights how the main core of the globin fold is capable of becoming fully native efficiently, on the sub-millisecond time scale.

High-resolution conformation and backbone dynamics of a soluble aggregate of apomyoglobin119.

The structure and dynamics of soluble misfolded aggregates are poorly understood, despite their importance in protein science and disease. Water-soluble self-associated species that do not become insoluble over time are invaluable tools for high-resolution conformational studies aimed at dissecting the determinants of self-association. Here, we characterize the soluble model aggregate apomyoglobin(119) (apoMb(119)), generated upon truncating the residues corresponding to the C-terminal helix of sperm whale apomyoglobin. The secondary structure and backbone dynamics of apoMb(119), determined by multidimensional NMR at pH 6.0, reveal the presence of an N-terminal slow-tumbling core and a highly disordered flexible C-terminus displaying residual helicity and large-amplitude backbone motions on the picosecond-to-nanosecond timescale. The backbone of the apoMb(119) aggregate assumes progressively increased mobility as residues get further removed from the nonpolar core and closer to the more hydrophilic C-terminal end. This structural motif establishes a useful paradigm for the topology of soluble misfolded protein aggregates in aqueous solution in the absence of stabilizing additives. The partially helical and flexible C-terminus of apoMb(119)'s aggregate is in interesting contrast with the amyloid-related globulomers, which display dangling ends rich in ß-strand. Finally, we investigate how a molecular chaperone, the substrate-binding domain of DnaK, interferes with apoMb(119)'s aggregation.

Effects of epilepsy and antiepileptic drugs on nitric oxide, lipid peroxidation and xanthine oxidase system in children with idiopathic epilepsy.

The aim of this study is to investigate the effects of epilepsy, valproic acid and oxcarbazepine on nitric oxide levels, lipid peroxidation and xanthine oxidase levels in newly diagnosed epileptic children and healthy controls. A total of 49 patients with newly diagnosed idiopathic epilepsy and 15 healthy children were enrolled in this study. Of these 49 patients, 16 children were treated with valproate and 16 treated with oxcarbazepine. Nitric oxide, malondialdehyde and xanthine oxidase levels prior to antiepileptic drug therapy were measured in the serum. Blood samples were drawn before antiepileptic drug therapy and after 3 and 6 months of the antiepileptic drug treatment. Nitric oxide levels were statistically higher in the newly diagnosed epileptic patients. In oxcarbazepine group, the nitric oxide and malondialdehyde levels were found to be decreased. No statistically significant differences were noted in nitric oxide, malondialdehyde and xanthine oxidase levels in valproic acid treated group. Oxcarbazepine which is a frequently used new antiepileptic drug in childhood epilepsy may modify nitric oxide levels and lipid peroxidation. These results suggest that decreased lipid peroxidation would play a role in the mechanism of antiepileptic effects by oxcarbazepine treatment.

Neonate with meroacrania: radiological findings and review of the literature.

Acrania is a developmental abnormality characterized by a partial or complete absence of calvaria with complete but abnormal development of brain tissue. Acrania is a relatively common malformation and affects about 1 in 1000 newborns. Meroacrania refers to absence of the cranium with the exception of the occipital bone. Brain stem and cerebellum develop normally, but cerebral parenchyma tissue is covered with a thin membrane and severely dysmorphic supratentorial brain is also seen. The other system findings are normal. Magnetic resonance imaging findings of one neonate with meroacrania have been reported in medical literature. Other radiographic and computed tomography findings have not yet been reported. We report a female neonate with meroacrania with discussion of etiology, pathogenesis, radiological findings, and differential diagnosis.

Nonrandom distribution of intramolecular contacts in native single-domain proteins.

The interplay of short- and long-range interactions in protein structure and folding is poorly understood. This study focuses on the distribution of intramolecular contacts across different regions of the polypeptide chain in soluble single-domain proteins. We show that while the average number of intramolecular interactions per residue is similar across all regions of the sequence, the interaction counterparts are distributed nonrandomly. Two types of proteins are observed. The first class comprises structures that have the majority of their intramolecular contacts linking amino acids within the same region of the sequence (i.e., N-/C-terminal or intermediate portion of the chain). A second smaller class includes proteins that have extensive contacts between the N and C termini. Such extensive interactions involve primarily distal beta-strands and are detected via the NCR parameter, a descriptor of the number of contacts with interaction counterparts in specific regions of the sequence. In summary, the majority of single-domain proteins (first class) is dominated by short-range interactions between contiguous elements of secondary structure and has only sparse contacts among the N and C termini. This finding defies the common assumption that the chain termini, often spatially close in folded proteins, have to participate in a large number of mutual interactions. Finally, our results suggest that the C-terminal region of Class 2 proteins may be particularly effective at promoting folding upon completion of protein biosynthesis in the cell.

A prospective study of etiology of childhood acute bacterial meningitis, Turkey.

Determination of the etiology of bacterial meningitis and estimating cost of disease are important in guiding vaccination policies. To determine the incidence and etiology of meningitis in Turkey, cerebrospinal fluid (CSF) samples were obtained prospectively from children (1 month-17 years of age) with a clinical diagnosis of acute bacterial meningitis. Multiplex PCR was used to detect DNA evidence of Streptococcus pneumoniae, Haemophilus influenzae type b (Hib), and Neisseria meningitidis. In total, 408 CSF samples were collected, and bacterial etiology was determined in 243 cases; N. meningitidis was detected in 56.5%, S. pneumoniae in 22.5%, and Hib in 20.5% of the PCR-positive samples. Among N. meningitidis-positive CSF samples, 42.7%, 31.1%, 2.2%, and 0.7% belonged to serogroups W-135, B, Y, and A, respectively. This study highlights the emergence of serogroup W-135 disease in Turkey and concludes that vaccines to prevent meningococcal disease in this region must provide reliable protection against this serogroup.

Residue-specific contact order and contact breadth in single-domain proteins: implications for folding as a function of chain elongation.

Cotranslational protein misfolding and aggregation are often responsible for inclusion body formation during in vivo protein expression. This study addresses the relations between protein folding/misfolding and the distribution of intramolecular interactions across different regions of the polypeptide chain in soluble single-domain proteins. The sequence regions examined here include the C terminus, which is synthesized last in the cell. Emphasis is placed on two parameters reporting on short- and long-range interactions, i.e., residue-specific contact order (RCO) and a new descriptor of intramolecular protein interaction networks denoted as residue-specific contact breadth (RCB). RCB illustrates the average spread in sequence of the residues serving as interaction counterparts. We show that both RCO and RCB are maximized at the chain termini for a large fraction of single-domain soluble proteins. A direct implication of this result is that the C terminus of the polypeptide chain, which is synthesized last during ribosome-assisted translation, plays a key role in the generation of native-like structure by establishing long-range interactions and generating contacts with interaction counterparts widely distributed across the sequence. Comparison of our computational predictions with the experimental behavior of selected proteins shows that the presence and absence of large RCO and RCB at the chain termini correlates with the protein's ability to properly fold either after the C terminus has been synthesized or during chain elongation, respectively.

Nonnative helical motif in a chaperone-bound protein fragment.

The effect of cotranslationally active chaperones on the conformation of incomplete protein chains is poorly understood. The secondary structure of a 77-residue chaperone-bound N-terminal protein fragment corresponding to the first five helices (A-E) of apomyoglobin (apoMb(1-77)) is investigated here at the residue-specific level by multidimensional NMR. The substrate-binding domain of DnaK, DnaK-beta, is employed as a chaperone model. By taking advantage of the improved spectral quality resulting from chaperone deuteration, we find that DnaK-beta-bound apoMb(1-77) displays a region of nonnative helicity at residues away from the main chaperone binding site. The nonnative structural motif comprises portions of the native D and E helices and has similar characteristics to the reported nonnative DE helical region of acid-unfolded full-length apoMb. Upon incorporation of the missing C-terminal amino acids, a structural kink develops between residues 56 and 57, and two separate native D and E helices are generated. This work highlights, for the first time to our knowledge, the presence of a nonnative helical motif in a large chaperone-bound protein fragment under physiologically relevant solution conditions.

Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin.

Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent Delta G degrees (UN(w))=-3.1+/-0.3 kcal mol(-1); m(UN)=-1.7 kcal mol(-1) M(-1)] and predominant alpha-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (beta(I)=0.9+/-0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.

Age-specific seroprevalence of serogroup C meningococcal serum bactericidal antibody activity and serogroup A, C, W135 and Y-specific IgG concentrations in the Turkish population during 2005.

Like many other developing countries; there is no accurate information about the antibody levels against Neisseria meningitidis in Turkey. We collected serum samples from four health centers located in different geographic regions and stratified according to age in order to obtain a baseline seroprevalence of protective antibodies to meningococcal serogroup C and provide data on seroprevalence of IgG antibodies to serogroups A, C, W135 and Y. Sera were tested for serum bactericidal antibodies (SBA) to serogroup C meningococci using rabbit serum as the complement source and by a bead based assay for serogroup A, C, W135 and Y-specific IgG. It was observed that 30% and 12% of individuals within the study population had SBA titers of > or =8 and > or =128, respectively. Overall; at least 70% of the population are susceptible (SBA titer <8) to meningococcal serogroup C disease. The rate of susceptibility was highest in infants aged 7-12 months and young children (1-4 years). Regardless of age, for serogroup A, C, W135 and Y, 60.5%, 27.2%, 12.3% and 19.2% of subjects, respectively, had serogroup-specific IgG concentrations > or =2 microg/mL. These data highlight that a large proportion of the Turkish population are susceptible to serogroups C, W135 and Y and should be considered, along with serogroup-specific disease incidence data, in future decisions on possible meningococcal vaccination programmes.

Binding specificity of an alpha-helical protein sequence to a full-length Hsp70 chaperone and its minimal substrate-binding domain.

Hsp70 chaperones are involved in the prevention of misfolding, and possibly the folding, of newly synthesized proteins. The members of this chaperone family are capable of interacting with polypeptide chains both co- and posttranslationally, but it is currently not clear how different structural domains of the chaperone affect binding specificity. We explored the interactions between the bacterial Hsp70, DnaK, and the sequence of a model all-alpha-helical globin (apoMb) by cellulose-bound peptide scanning. The binding specificity of the full-length chaperone was compared with that of its minimal substrate-binding domain, DnaK-beta. Six specific chaperone binding sites evenly distributed along the apoMb sequence were identified. Binding site locations are identical for the full-length chaperone and its substrate-binding domain, but relative affinities differ. The binding specificity of DnaK-beta is only slightly decreased relative to that of full-length DnaK. DnaK's binding motif is known to comprise hydrophobic regions flanked by positively charged residues. We found that the simple fractional mean buried area correlates well with Hsp70's binding site locations along the apoMb sequence. In order to further characterize the properties of the minimal binding host, the stability of DnaK-beta upon chemical denaturation by urea and protons was investigated. Urea unfolding titrations yielded an apparent folding DeltaG degrees of 3.1 +/- 0.9 kcal mol-1 and an m value of 1.7 +/- 0.4 kcal mol-1 M-1.

Secondary structure mapping of DnaK-bound protein fragments: chain helicity and local helix unwinding at the binding site.

Little is known about polypeptide conformation and folding in the presence of molecular chaperones participating in protein biosynthesis. In vitro studies on chaperone-substrate complexes have been mostly carried out with small peptide ligands. However, the technical challenges associated with either competing aggregation or spectroscopically unfavorable size and exchange rates have typically prevented analysis of larger substrates. Here, we report the high-resolution secondary structure of relatively large N-terminal protein fragments bound to the substrate-binding domain of the cotranslationally active chaperone DnaK. The all-alpha-helical protein apomyoglobin (apoMb), bearing the ubiquitous globin fold, has been chosen as a model substrate. On the basis of NMR secondary chemical shift analysis, we identify, for the first time, weak helical content (similar to that found in the chemically unfolded full-length protein) for the assigned residues of the chaperone-bound chain away from the chaperone binding sites. In contrast, we found that the residues corresponding to the strongest specific binding site for DnaK, examined via a short 13-mer apoMb peptide fragment matching the binding site sequence, display highly reduced helical content in their chaperone-bound form. Given that the free state of the peptide is weakly helical in isolation, we conclude that the substrate residues corresponding to the chaperone binding site undergo helix unwinding upon chaperone binding.

Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution.

The biophysical characterization of nonfunctional protein aggregates at physiologically relevant temperatures is much needed to gain deeper insights into the kinetic and thermodynamic relationships between protein folding and misfolding. Dynamic and static laser light scattering have been employed for the detection and detailed characterization of apomyoglobin (apoMb) soluble aggregates populated at room temperature upon dissolving the purified protein in buffer at pH 6.0, both in the presence and absence of high concentrations of urea. Unlike the beta-sheet self-associated aggregates previously reported for this protein at high temperatures, the soluble aggregates detected here have either alpha-helical or random coil secondary structure, depending on solvent and solution conditions. Hydrodynamic diameters range from 80 to 130 nm, with semiflexible chain-like morphology. The combined use of low pH and high urea concentration leads to structural unfolding and complete elimination of the large aggregates. Even upon starting from this virtually monomeric unfolded state, however, protein refolding leads to the formation of severely self-associated species with native-like secondary structure. Under these conditions, kinetic apoMb refolding proceeds via two parallel routes: one leading to native monomer, and the other leading to a misfolded and heavily self-associated state bearing native-like secondary structure.

Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain.

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-beta. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-beta, however, formation of the original self-associated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-beta-bound state, which becomes folding-competent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently outcompetes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.

The burial of solvent-accessible surface area is a predictor of polypeptide folding and misfolding as a function of chain elongation.

The hydrophobic effect is a major driving force in all chemical and biological events involving chain collapse in aqueous solution. Here, we show that the burial of nonpolar solvent-accessible surface area (NSASA) is a powerful criterion to predict the folding and misfolding behavior of small single-domain proteins as a function of chain elongation. This bears fundamental implications for co- and post-translational protein folding in the cell and for understanding the interplay between noncovalent interactions and formation of native-like structure and topology. Comparison between the fraction of NSASA in fully unfolded and folded elongating chains shows that efficient burial of nonpolar surface area is preferentially achieved only when the polypeptide chain is almost complete. This effect has no preferential vectorial character in that it is present upon elongation from both the N and C termini. For incomplete chains that do not have the ability to fold and bury nonpolar surface intramolecularly, the overall hydrophobic nature of the polypeptide chain (expressed as FBA, i.e., fractional buried surface area per residue) dictates the tendency toward misfolding and self-association. N-terminal chains characterized by FBA exceeding 0.73 are likely to misfold and aggregate, if unable to fold intramolecularly.

Structure-based prediction of potential binding and nonbinding peptides to HIV-1 protease.

HIV-1 protease is a major drug target against AIDS as it permits viral maturation by processing the gag and pol polyproteins of the virus. The cleavage sites in these polyproteins do not have obvious sequence homology or a binding motif and the specificity of the protease is not easily determined. We used various threading approaches, together with the crystal structures of substrate complexes which served as template structures, to study the substrate specificity of HIV-1 protease with the aim of obtaining a better differentiation between binding and nonbinding sequences. The predictions from threading improved when distance-dependent interaction energy functions were used instead of contact matrices. To rank the peptides and properly account for the peptide's conformation in the total energy, the results from using short-range potentials on multiple template structures were averaged. Finally, a dynamic threading approach is introduced which is potentially useful for cases when there is only one template structure available. The conformational energy of the peptide-especially the term accounting for the side chains-was found to be important in differentiating between binding and nonbinding sequences. Hence, the substrate specificity, and thus the ability of the virus to mature, is affected by the compatibility of the substrate peptide to fit within the limited conformational space of the active site groove.

Cooperative fluctuations of unliganded and substrate-bound HIV-1 protease: a structure-based analysis on a variety of conformations from crystallography and molecular dynamics simulations.

The dynamics of HIV-1 protease, both in unliganded and substrate-bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV-1 protease are conserved throughout the MD simulations. The flaps and 40's loop of the unliganded HIV-1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate-bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV-1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool.