New insights into a spectrum of developmental malformations related to mTOR dysregulations: challenges and perspectives.

In recent years the role of the mammalian target of rapamycin (mTOR) pathway has emerged as crucial for normal cortical development. Therefore, it is not surprising that aberrant activation of mTOR is associated with developmental malformations and epileptogenesis. A broad spectrum of malformations of cortical development, such as focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC), have been linked to either germline or somatic mutations in mTOR pathway-related genes, commonly summarised under the umbrella term 'mTORopathies'. However, there are still a number of unanswered questions regarding the involvement of mTOR in the pathophysiology of these abnormalities. Therefore, a monogenetic disease, such as TSC, can be more easily applied as a model to study the mechanisms of epileptogenesis and identify potential new targets of therapy. Developmental neuropathology and genetics demonstrate that FCD IIb and hemimegalencephaly are the same diseases. Constitutive activation of mTOR signalling represents a shared pathogenic mechanism in a group of developmental malformations that have histopathological and clinical features in common, such as epilepsy, autism and other comorbidities. We seek to understand the effect of mTOR dysregulation in a developing cortex with the propensity to generate seizures as well as the aftermath of the surrounding environment, including the white matter.

Time delay in the swing equation: A variety of bifurcations.

The present paper addresses the swing equation with additional delayed damping as an example for pendulumlike systems. In this context, it is proved that recurring sub- and supercritical Hopf bifurcations occur if time delay is increased. To this end, a general formula for the first Lyapunov coefficient in second order systems with additional delayed damping and delay-free nonlinearity is given. Insofar, the paper extends the results about the stability switching of equilibria in linear time delay systems from Cooke and Grossman. In addition to the analytical results, periodic solutions are numerically dealt with. The numerical results demonstrate how a variety of qualitative behaviors are generated in the simple swing equation by only introducing time delay in a damping term.

Sheathless coupling of microchip electrophoresis to ESI-MS utilising an integrated photo polymerised membrane for electric contacting.

In this article, we present a novel approach for the sheathless coupling of microchip electrophoresis (MCE) with electrospray mass spectrometry (ESI-MS). The key element is an ion-conductive hydrogel membrane, placed between the separation channel and an adjacent microfluidic supporting channel, contacted via platinum electrodes. This solves the persistent challenge in hyphenation of mass spectrometry to chip electrophoresis, to ensure a reliable electrical connection at the end of the electrophoresis channel without sacrificing separation performance and sensitivity. Stable electric contacting is achieved via a Y-shaped supporting channel structure, separated from the main channel by a photo polymerised, ion permeable hydrogel membrane. Thus, the potential gradient required for performing electrophoretic separations can be generated while simultaneously preventing gas formation due to electrolysis. In contrast to conventional make-up or sheathflow approaches, sample dilution is also avoided. Rapid prototyping allowed the study of different chip-based approaches, i.e. sheathless, open sheathflow and electrode support channel designs, for coupling MCE to ESI-MS. The performance was evaluated with fluorescence microscopy and mass spectrometric detection. The obtained results revealed that the detection sensitivity obtained in such Y-channel chips with integrated hydrogel membranes was superior because sample dilution or loss was prevented. Furthermore, band broadening is reduced compared to similar open structures without a membrane.

Expression of microRNAs miR21, miR146a, and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA-derived cell cultures.

Tuberous sclerosis complex (TSC) is a genetic disease presenting with multiple neurological symptoms including epilepsy, mental retardation, and autism. Abnormal activation of various inflammatory pathways has been observed in astrocytes in brain lesions associated with TSC. Increasing evidence supports the involvement of microRNAs in the regulation of astrocyte-mediated inflammatory response. To study the role of inflammation-related microRNAs in TSC, we employed real-time PCR and in situ hybridization to characterize the expression of miR21, miR146a, and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). We observed an increased expression of miR21, miR146a, and miR155 in TSC tubers compared with control and perituberal brain tissue. Expression was localized in dysmorphic neurons, giant cells, and reactive astrocytes and positively correlated with IL-1ß expression. In addition, cultured human astrocytes and SEGA-derived cell cultures were used to study the regulation of the expression of these miRNAs in response to the proinflammatory cytokine IL-1ß and to evaluate the effects of overexpression or knockdown of miR21, miR146a, and miR155 on inflammatory signaling. IL-1ß stimulation of cultured glial cells strongly induced intracellular miR21, miR146a, and miR155 expression, as well as miR146a extracellular release. IL-1ß signaling was differentially modulated by overexpression of miR155 or miR146a, which resulted in pro- or anti-inflammatory effects, respectively. This study provides supportive evidence that inflammation-related microRNAs play a role in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte-mediated inflammatory response, with miR146a as most interesting anti-inflammatory therapeutic candidate.

Dysregulation of the (immuno)proteasome pathway in malformations of cortical development.

BACKGROUND: The proteasome is a multisubunit enzyme complex involved in protein degradation, which is essential for many cellular processes. During inflammation, the constitutive subunits are replaced by their inducible counterparts, resulting in the formation of the immunoproteasome. METHODS: We investigated the expression pattern of constitutive (ß1, ß5) and immunoproteasome (ß1i, ß5i) subunits using immunohistochemistry in malformations of cortical development (MCD; focal cortical dysplasia (FCD) IIa and b, cortical tubers from patients with tuberous sclerosis complex (TSC), and mild MCD (mMCD)). Glial cells in culture were used to elucidate the mechanisms regulating immunoproteasome subunit expression. RESULTS: Increased expression was observed in both FCD II and TSC; ß1, ß1i, ß5, and ß5i were detected (within cytosol and nucleus) in dysmorphic neurons, balloon/giant cells, and reactive astrocytes. Glial and neuronal nuclear expression positively correlated with seizure frequency. Positive correlation was also observed between the glial expression of constitutive and immunoproteasome subunits and IL-1ß. Accordingly, the proteasome subunit expression was modulated by IL-1ß in human astrocytes in vitro. Expression of both constitutive and immunoproteasome subunits in FCD II-derived astroglial cultures was negatively regulated by treatment with the immunomodulatory drug rapamycin (inhibitor of the mammalian target of rapamycin (mTOR) pathway, which is activated in both TSC and FCD II). CONCLUSIONS: These observations support the dysregulation of the proteasome system in both FCD and TSC and provide new insights on the mechanism of regulation the (immuno)proteasome in astrocytes and the molecular links between inflammation, mTOR activation, and epilepsy.

Detection of radiation-induced lung injury using hyperpolarized (13)C magnetic resonance spectroscopy and imaging.

Radiation-induced lung injury limits radiotherapy of thoracic cancers. Detection of radiation pneumonitis associated with early radiation-induced lung injury (2-4 weeks postirradiation) may provide an opportunity to adjust treatment, before the onset of acute pneumonitis and/or irreversible fibrosis. In this study, localized magnetic resonance (MR) spectroscopy and imaging of hyperpolarized (13)C-pyruvate (pyruvate) and (13)C-lactate (lactate) were performed in the thorax and kidney regions of rats 2 weeks following whole-thorax irradiation (14 Gy). Lactate-to-pyruvate signal ratio was observed to increase by 110% (P < 0.01), 57% (P < 0.02), and 107% (P < 0.01), respectively, in the thorax, lung, and heart tissues of the radiated rats compared with healthy age-matched rats. This was consistent with lung inflammation confirmed using cell micrographs of bronchioalveolar lavage specimens and decreases in arterial oxygen partial pressure (paO2), indicative of hypoxia. No statistically significant difference was observed in either lactate-to-pyruvate signal ratios in the kidney region (P = 0.50) between the healthy (0.215 ± 0.100) and radiated cohorts (0.215 ± 0.054) or in blood lactate levels (P = 0.69) in the healthy (1.255 ± 0.247 mmol/L) and the radiated cohorts (1.325 ± 0.214 mmol/L), confirming that the injury is localized to the thorax. This work demonstrates the feasibility of hyperpolarized (13)C metabolic MR spectroscopy and imaging for detection of early radiation-induced lung injury.

[Suicidal gas embolism in hospital]. / Suizidale Gasembolie im Krankenhaus.

Air embolisms are a common entity seen in clinical practice after traumatic or iatrogenic events. Cases of a suicidally induced air embolism are rare. The connectivity of air-carrying and fluid-carrying tubing systems allow large gas volumes to be infused in a short period of time, usually with fatal outcome. In such cases, the use of computed tomography prior to autopsy is mandatory and provides a comprehensive visualization of introduced gas volumes. We present an unusual case of a suicidal gas embolism, in which a man in hospital connected a tube supplying oxygen to a venous catheter.

Cooperativity between the J and S elements of class II major histocompatibility complex genes as enhancers in normal and class II-negative patient and mutant B cell lines.

The class II major histocompatibility complex genes all contain in their proximal promoters three cis-elements called S, X, and Y that are conserved in both sequence and position, and a fourth element, J, conserved in sequence but not in position. J, X, and Y and, to some extent, S, have been shown to be functionally important in regulation of expression of these genes. In the present study, a protein factor that binds cooperatively to the S plus J elements of the promoter of the class II major histocompatibility complex gene DPA has been detected. Moreover, functional cooperativity between S and J in activation of the enhancerless -40 interferon-beta (-40 IFN-beta) promoter has been demonstrated. Finally, the latter assay appears to subdivide complementation group A of class II negative human B cell lines that includes both mutants generated in vitro and cells from patients with the bare lymphocyte syndrome (type II). In three of these cell lines, the enhancerless -40 IFN-beta promoter containing the S plus J elements was functionally active, while in the others it was inactive.

Differences in SMN1 allele frequencies among ethnic groups within North America.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common inherited lethal disease of children. Various genetic deletions involving the bi-allelic loss of SMN1 exon 7 are reported to account for 94% of affected individuals. Published literature places the carrier frequency for SMN1 mutations between 1 in 25 and 1 in 50 in the general population. Although SMA is considered to be a pan-ethnic disease, carrier frequencies for many ethnicities, including most ethnic groups in North America, are unknown. OBJECTIVES AND METHODS: To provide an accurate assessment of SMN1 mutation carrier frequencies in African American, Ashkenazi Jewish, Asian, Caucasian, and Hispanic populations, more than 1000 specimens in each ethnic group were tested using a clinically validated, quantitative real-time polymerase chain reaction (PCR) assay that measures exon 7 copy number. RESULTS: The observed one-copy genotype frequency was 1 in 37 (2.7%) in Caucasian, 1 in 46 (2.2%) in Ashkenazi Jew, 1 in 56 (1.8%) in Asian, 1 in 91 (1.1%) in African American, and 1 in 125 (0.8%) in Hispanic specimens. Additionally, an unusually high frequency of alleles with multiple copies of SMN1 was identified in the African American group (27% compared to 3.3-8.1%). This latter finding has clinical implications for providing accurate adjusted genetic risk assessments to the African American population. CONCLUSIONS: Differences in the frequency of SMA carriers were significant among several ethnic groups. This study provides an accurate assessment of allele frequencies and estimates of adjusted genetic risk that were previously unavailable to clinicians and patients considering testing.

A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein.

A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene.

Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral.

BACKGROUND: Genetic testing for hereditary cancer syndromes contributes to the medical management of patients who may be at increased risk of one or more cancers. BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer is one such widely used test. However, clinical testing methods with high sensitivity for deleterious mutations in these genes also detect many unclassified variants, primarily missense substitutions. METHODS: We developed an extension of the Grantham difference, called A-GVGD, to score missense substitutions against the range of variation present at their position in a multiple sequence alignment. Combining two methods, co-occurrence of unclassified variants with clearly deleterious mutations and A-GVGD, we analysed most of the missense substitutions observed in BRCA1. RESULTS: A-GVGD was able to resolve known neutral and deleterious missense substitutions into distinct sets. Additionally, eight previously unclassified BRCA1 missense substitutions observed in trans with one or more deleterious mutations, and within the cross-species range of variation observed at their position in the protein, are now classified as neutral. DISCUSSION: The methods combined here can classify as neutral about 50% of missense substitutions that have been observed with two or more clearly deleterious mutations. Furthermore, odds ratios estimated for sets of substitutions grouped by A-GVGD scores are consistent with the hypothesis that most unclassified substitutions that are within the cross-species range of variation at their position in BRCA1 are also neutral. For most of these, clinical reclassification will require integrated application of other methods such as pooled family histories, segregation analysis, or validated functional assay.

Design of field-cycled magnetic resonance systems for small animal imaging.

This paper presents a design study for a field-cycled magnetic resonance imaging (MRI) system directed at small animal imaging applications. A field-cycled MRI system is different from a conventional MRI system in that it uses two separate and dynamically controllable magnetic fields. A strong magnetic field is used to polarize the object, and a relatively weak magnetic field is used during signal acquisition. The potential benefits of field-cycled MRI are described. The theoretical dependences of field-cycled MRI performance on system design are introduced and investigated. Electromagnetic, mechanical and thermal performances of the system were considered in this design study. A system design for imaging 10 cm diameter objects is presented as an example, capable of producing high-duty-cycle polarizing magnetic fields of 0.5 T and readout magnetic fields corresponding to a proton Larmor frequency of 5 MHz. The specifications of the final design are presented along with its expected electromagnetic and thermal performance.

Promoter-Specific Hypomethylation Correlates with IL-1ß Overexpression in Tuberous Sclerosis Complex (TSC).

In tuberous sclerosis complex (TSC), overexpression of numerous genes associated with inflammation has been observed. Among different proinflammatory cytokines, interleukin-1ß (IL-1ß) has been shown to be significantly involved in epileptogenesis and maintenance of seizures. Recent evidence indicates that IL-1ß gene expression can be regulated by DNA methylation of its promoter. In the present study, we hypothesized that hypomethylation in the promoter region of the IL-1ß gene may underlie its overexpression observed in TSC brain tissue. Bisulfite sequencing was used to study the methylation status of the promoter region of the IL-1ß gene in TSC and control samples. We identified hypomethylation in the promoter region of the IL-1ß gene in TSC samples. IL-1ß is overexpressed in tubers, and gene expression is correlated with promoter hypomethylation at CpG and non-CpG sites. Our results provide the first evidence of epigenetic modulation of the IL-1ß signaling in TSC. Thus, strategies that target epigenetic alterations could offer new therapeutic avenues to control the persistent activation of interleukin-1ß-mediated inflammatory signaling in TSC brain.

Application of haplotype pair analysis for the identification of hemizygous loci.

An expectation maximisation based prediction algorithm was created to identify unusual haplotypes in patient samples that may be caused by small intragenic deletions. In this approach, unphased SNP genotypes are compared to pairs of canonical haplotypes to identify potentially hemizygous regions. This method was successfully applied to identify five deletions in the 3' region of BRCA1.

The reproducibility of ultrasound bone measures in a triethnic population of pregnant adolescents and adult women.

We used bone ultrasound technology with its measurement of attenuation (broadband ultrasound attenuation [BUA] as dB/MHz) and sound velocity (speed of sound as m/s) for assessing the quantitative ultrasound index (QUI) summary measure in a triethnic population of 280 pregnant women. The study purpose was to describe the reproducibility of the ultrasound technology and determine if the correlations of age, weight, and ethnicity with the bone status measures in this population are consistent with the correlations of age, weight, and ethnicity that have been reported with other technologies that measure bone mass. We evaluated the first 280 women enrolled in our longitudinal study of lead turnover from maternal bone during pregnancy and lactation. Enrollees were pregnant, aged 12-29 years, and self-classified as black, white, or Hispanic. Bone ultrasound was measured twice at entry to prenatal care, which, on average, was at 14 weeks gestation. Reproducibility was described with intraclass correlations and the standard error of measurement. Age, weight, and ethnicity were associated with bone status measures using Spearman correlations and generalized linear models. The reproducibility of the summary bone measure, QUI, was high (96-97%). Variation in age and ethnicity did not alter reproducibility; however, the reproducibility of the attenuation measure (BUA as dB/MHz) lessened with increasing weight, declining from 95% to 89%. Since this attenuation is included in the summary QUI measure, there was a slight, and nonsignificant, decline in QUI reproducibility (from 97% to 96%) as women increased in size. There were no statistically significant differences in mean bone ultrasound measures according to age, where ages ranged from 12-29 years. Women who categorized themselves as black had, on average, an 8.5% greater QUI than did women who classified themselves as Hispanic or white. There were no significant pair-wise differences in mean ultrasound measures of bone between women classifying themselves as Hispanic or white. The use of ultrasound is a highly reproducible measure to assess bone characteristics in a population of pregnant adolescent and young adult women and its summary measure of bone mass is correlated with ethnic as well as body size characteristics.

IGF-I, osteocalcin, and bone change in pregnant normotensive and pre-eclamptic women.

Pre-eclampsia is a pregnancy disorder of uncertain etiology that affects 5-10% of all pregnancies, with symptoms typically presenting around or after 20 wk gestation. We hypothesized that IGF-I, osteocalcin, and bone loss would be different among women with pre-eclampsia compared with normotensive pregnant women. There were 962 pregnant healthy women, aged 12-35, who were assessed at entry to care, at 28 wk, and at delivery for osteocalcin and IGF-I concentrations. Bone ultrasound was measured at entry to care and at 6 wk postpartum, whereas bone mineral density was measured by dual x-ray densitometry at delivery. There were 64 women (6.7%) among the women being followed who developed pre-eclampsia. In women with pre-eclampsia, IGF-I concentrations were 74% greater in the third trimester compared with the first trimester, whereas there was little change in osteocalcin concentrations. In contrast, normotensive women had an average increase of 43% in IGF-I concentrations accompanied by a 63% decline in osteocalcin concentrations. In women with pre-eclampsia, IGF-I and osteocalcin concentrations were significantly correlated (r = 0.48 and 0.43) at both the first and third trimester time points, but only in the third trimester among normotensive women (r = 0.27). The bone change difference between the two groups was not statistically significant. Women with pre-eclampsia appear to have an exaggerated IGF-I responsiveness compared with women who are normotensive; however, the strong correlation between IGF-I and osteocalcin in women with pre-eclampsia suggests that the IGF-I is able to retain its role as a local regulator of bone remodeling, as indicated by the osteocalcin concentrations.

Folic acid: influence on the outcome of pregnancy.

The periconceptional use of folic acid-containing supplements reduces the first occurrence, as well as the recurrence, of neural tube defects. Women of populations in which adverse pregnancy outcomes are prevalent often consume diets that contain a low density of vitamins and minerals, including folate. Folate intake may need to be sustained after complete closure of the neural tube to decrease the risk of other poor pregnancy outcomes. A central feature of embryonic and fetal development is widespread cell division; folate is central because of its role in nucleic acid synthesis. During gestation, marginal folate nutriture can impair cellular growth and replication in the fetus or placenta. Folate deficiency can occur because dietary folate intake is low or because the metabolic requirement for folate is increased by a particular genetic defect or defects. During pregnancy, low concentrations of dietary and circulating folate are associated with increased risks of preterm delivery, infant low birth weight, and fetal growth retardation. A metabolic effect of folate deficiency is an elevation of blood homocysteine. Likewise, the presence of maternal homocysteine concentrations have been associated both with increased habitual spontaneous abortion and pregnancy complications (eg, placental abruption and preeclampsia), which increase the risk of poor pregnancy outcome and of decreased birth weight and gestation duration.

Anemia and iron-deficiency anemia: compilation of data on pregnancy outcome.

Anemia diagnosed early in pregnancy is associated with increased risks of low birth weight and preterm delivery. In several studies, the association between anemia and outcomes reversed direction during the third trimester; maternal anemia was no longer a risk factor for poor pregnancy outcomes. Camden study data were used to examine the probable cause of this observation. Maternal iron-deficiency anemia, diagnosed at entry to prenatal care, was associated with low dietary energy and iron, inadequate gestational gain, and twofold or greater increases in the risks of preterm delivery and low birth weight. During the third trimester, these associations (except with inadequate gestational gain) were no longer present. This reversal of risk status may be attributable to the poor predictive value of anemia and iron deficiency tests during the third trimester. However, the relationship between poor diet (with inadequate iron intake) and increased likelihood of preterm delivery persisted during the third trimester.

Leptin and maternal growth during adolescent pregnancy.

BACKGROUND: Maternal growth on the basis of knee height occurs in nearly 50% of pregnant teenagers and is associated with greater gestational weight gain and accrual of subcutaneous fat in the mother but lower fetal growth compared with nongrowing teenagers and mature pregnant women. OBJECTIVE: The objective of this study was to determine whether leptin is a biomarker for continued maternal growth. DESIGN: Leptin concentrations were measured in 162 growing and nongrowing teenage gravidas (aged 5-fold, fetal growth restriction increased >6-fold, and infant birth weight decreased by approximately 200 g. Gravidas who developed pregnancy-induced hypertension showed a different pattern-higher leptin concentrations at entry and week 28, no difference in the leptin surge, and no postpartum difference in leptin concentration. CONCLUSION: A leptin surge by week 28 appears to mark reduced mobilization of maternal fat stores that is associated with maternal growth on the basis of knee height during adolescent pregnancy.

Maternal growth during pregnancy and decreased infant birth weight.

We used stature and measurement of knee height to measure continued maternal growth during adolescent pregnancy in a sample of young gravidas (primigravidas and multiparas) and mature pregnant control subjects. Growth during pregnancy has been masked by a tendency of all gravidas to shrink while pregnant (approximately 0.5 cm over 6 mo of observation). Consequently, growth of many adolescent gravidas has not been clinically apparent. There was no effect on maternal growth during a first pregnancy in adolescence but this may be a result of the relatively good prepregnant nutrition status of the young gravidas in developed countries. Maternal growth during pregnancy, however, is associated with significantly decreased (-282 g, p less than 0.05) birth weight for infants when maternal growth continues during a subsequent adolescent pregnancy. This observation is consistent with the hypothesized competition between the metabolic demands of the growing adolescent mother and the nutrient needs of her developing fetus.