[Developmental neurology - networked medicine and new perspectives]. / Entwicklungsneurologie ­ vernetzte Medizin und neue Perspektiven.

Developmental neurology is one of the major areas of neuropediatrics and is among other things (legally) responsible for monitoring the motor, cognitive and psychosocial development of all infants using standardized monitoring investigations. The special focus is on infants born at risk and/or due to premature birth before 32 weeks of gestation or a birth weight less than 1500 g. Early diagnosis of deviations from normal, age-related development is a prerequisite for early interventions, which may positively influence development and the long-term biopsychosocial prognosis of the patients. This article illustrates the available methods in developmental neurology with a focus on recent developments. Particular attention is paid to the predictive value of general movements (GM). The current development of markerless automated detection of spontaneous movements using conventional depth imaging cameras is demonstrated. Differences in spontaneous movements in infants at the age of 12 weeks are illustrated and discussed exemplified by three patients (healthy versus genetic syndrome versus cerebral palsy).

[Migraine in children and adolescents-brain and muscle? : Another example of why children are not small adults]. / Migräne im Kindes- und Jugendalter ­ Gehirn und Muskel? : Warum Kinder einmal mehr keine kleinen Erwachsenen sind.

Migraine as primary headache is a life-long disease which is relevant for the quality of life and is based on complex genetics. It often starts in childhood with symptoms typical for the specific age. These show different nuances compared to the migraine symptoms in adults, for example, regarding (bilateral/unilateral) localization of the acute migraine headache. Only over the course of years-during adolescence and young adulthood-do the more specific symptoms as defined by the International Classification of Headache Disorders (ICHD 3 beta) develop. In this article we focus on the clinical specifics of children and adolescents with migraine. We elaborately refer to the trigeminocervical complex (TCC) because it forms a conceptual bridge for the understanding of migraine, for psychoeducation, and for therapeutic options. We pragmatically discuss options and limits of treatments.

How does knowledge about spinal cord injury-related complications develop in subjects with spinal cord injury? A descriptive analysis in 214 patients.

STUDY DESIGN: Monocentric cohort study. OBJECTIVE: To investigate the acquisition of knowledge about spinal cord injury (SCI)-related complications in SCI patients. SETTING: Level 1 trauma center. METHODS: All patients with a traumatic or non-traumatic SCI were included in the study. Data were collected at admission, post-admission at 1 and 3 months and post-discharge at 6, 18 and 30 months. The discharge of all patients was between 3 and 6 months post-admission. Knowledge about pressure ulcers and bladder management was tested using the 'Knowledge' score. This score has a minimum and maximum of 0 and 20 points. To detect differences across the multiple time intervals, the Friedman test was used. Differences in the number of patients with poor (0-8), average (9-12) and good knowledge (13-20) between the different age classifications (age at injury) were calculated using a χ (2)-test. RESULTS: A total of 214 patients were included. At discharge subjects had increased their knowledge score to 11.2 compared with 5.4 on admission (P < 0.001). After 30 months, however, the mean score decreased to 10.8 points. At the time of discharge, the number of patients who achieved poor, average or good knowledge were 48 (22.4%), 65 (30.4%) and 101 (47.2%), respectively. Subjects of ∼50 years old and tetraplegics had better (P < 0.001) knowledge compared with subjects of ∼50 years old and paraplegics, respectively. CONCLUSION: In this study, less than 50% of SCI patients had good knowledge about bladder management and pressure ulcers after being discharged.

Atomic force spectroscopy evidence of non-specific adhesion of Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative periodontopathogen found within the subgingival plaque on the tooth surface. It is associated with localized aggressive periodontitis, a severe form of periodontitis in adolescents, and is the cause various extra-oral infections. The ability of this organism to tenaciously adhere to abiotic surfaces has been attributed to bundle-forming type IVb-like fimbriae whose major component is the fimbrial lower molecular weight protein (Flp). In this study the adhesion of purified Flp fimbriae isolated from A. actinomycetemcomitans to materials with different surface chemistries was measured using atomic force microscopy (AFM). The adhesion of Flp fimbriae to uncoated and saliva-coated tooth, hydroxylapatite, and glass surfaces was compared. Force data were used to quantify the magnitude of adhesion of the fimbriae and force-distance profiles were used to predict the mechanisms of adhesion. The results of this study confirm that non-specific interactions likely dominate the adhesion of these fimbriae to the surfaces used in this study. However, force data indicate that non-specific electrostatic interactions may be more significant under the conditions used in this study. Salivary coatings did affect both the hydrophobicity and adhesion of these fimbriae to the surfaces used in this study. Additionally, these data demonstrate the affect of salivary proteins on bacterial adhesion in the oral cavity.

Parameters for positive outcome of the in-hospital rehabilitation of spinal cord lesion patients: the Boberg Quality Score.

STUDY DESIGN: Monocentric Cohort Study. OBJECTIVE: Development of a multi-dimensional outcome score of the rehabilitation of spinal cord injury (SCI) patients. SETTING: Level 1 Trauma centre with large spinal unit. METHODS: During the rehabilitation of 161 patients with SCI between 2005 and 2007 following outcome parameters were assessed at different time points: functional status, pain, emotion, energy, sleep, social isolation, knowledge and self-management attitude. The results for these parameters were statistically evaluated with regard to different grades of paralysis and analyzed for socio-demographic influence factors. Correlation and factor analysis were then applied to evaluate the dependencies of the parameters and the dimensional structure of the applied score. RESULTS: For all factors a positive trend was found during the course of rehabilitation. Only the functional status is caused by spinal cord lesion. Age, sex and social isolation have no direct influence on the result with regard to functional status. For interactive dependencies of the parameters a three-dimensional structure was found: Success in the parameters, emotion, energy or social status, is independent of success in the parameter functional status. The result of the functional status is, next to the type of spinal injury, mainly influenced by the self-management attitude. The level of knowledge gained during rehabilitation is independent of the success in the other parameters. CONCLUSION: By the combination of functional, psychological and cognitive parameters into a result score it is possible to determine the quality of a rehabilitation process in its multiple dimensions and for different levels and grades of palsy. By this it is possible to analyze and compare the effectiveness and efficiency of rehabilitation on a national and international level. Longitudinal comparison of the long-term effects of rehabilitation after SCI is also possible, for example, in the course of follow-up examinations. SPONSORSHIP: Deutsche Gesetzliche Unfallversicherung (German Statutory Occupational Accident Insurance).

Intranasal insulin to improve developmental delay in children with 22q13 deletion syndrome: an exploratory clinical trial.

BACKGROUND: The 22q13 deletion syndrome (Phelan-McDermid syndrome) is characterised by a global developmental delay, absent or delayed speech, generalised hypotonia, autistic behaviour and characteristic phenotypic features. Intranasal insulin has been shown to improve declarative memory in healthy adult subjects and in patients with Alzheimer disease. AIMS: To assess if intranasal insulin is also able to improve the developmental delay in children with 22q13 deletion syndrome. METHODS: We performed exploratory clinical trials in six children with 22q13 deletion syndrome who received intranasal insulin over a period of 1 year. Short-term (during the first 6 weeks) and long-term effects (after 12 months of treatment) on motor skills, cognitive functions, or autonomous functions, speech and communication, emotional state, social behaviour, behavioural disorders, independence in daily living and education were assessed. RESULTS: The children showed marked short-term improvements in gross and fine motor activities, cognitive functions and educational level. Positive long-term effects were found for fine and gross motor activities, nonverbal communication, cognitive functions and autonomy. Possible side effects were found in one patient who displayed changes in balance, extreme sensitivity to touch and general loss of interest. One patient complained of intermittent nose bleeding. CONCLUSIONS: We conclude that long-term administration of intranasal insulin may benefit motor development, cognitive functions and spontaneous activity in children with 22q13 deletion syndrome.

Ultrasensitive determination of polycyclic aromatic compounds with atmospheric-pressure laser ionization as an interface for GC/MS.

Recently we introduced atmospheric pressure laser ionization (APLI) as a complementary ionization method for coupling LC-MS systems (HPLC and CEC), allowing ionization of nonpolar aromatic compounds via near-resonant two-photon excitation. In this paper, we demonstrate that APLI with the same source enclosure as for LC coupling is also suited for hyphenation of GC with atmospheric-pressure ionization mass spectrometry. This technique permits the qualitative and quantitative determination of aromatic compounds in an ultralow concentration range, as we show here with polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, and hetero-PAHs as examples. The outstanding sensitivity is demonstrated for chrysene, with a detection limit of 22 amol. Polar functional groups reduce the sensitivity, but after methylation or silylation, the analytes can also be determined very sensitively in complex matrixes, as is shown with 1-hydroxypyrene in urine.

Atmospheric-pressure laser ionization: a novel ionization method for liquid chromatography/mass spectrometry.

We report on the development of a new laser-ionization (LI) source operating at atmospheric pressure (AP) for liquid chromatography/mass spectrometry (LC/MS) applications. APLI is introduced as a powerful addition to existing AP ionization techniques, in particular atmospheric-pressure chemical ionization (APCI), electrospray ionization (ESI), and atmospheric pressure photoionization (APPI). Replacing the one-step VUV approach in APPI with step-wise two-photon ionization strongly enhances the selectivity of the ionization process. Furthermore, the photon flux during an ionization event is drastically increased over that of APPI, leading to very low detection limits. In addition, the APLI mechanism generally operates primarily directly on the analyte. This allows for very efficient ionization even of non-polar compounds such as polycyclic aromatic hydrocarbons (PAHs). The APLI source was characterized with a MicroMass Q-Tof Ultima II analyzer. Both the effluent of an HPLC column containing a number of PAHs (benzo[a]pyrene, fluoranthene, anthracene, fluorene) and samples from direct syringe injection were analyzed with respect to selectivity and sensitivity of the overall system. The liquid phase was vaporized by a conventional APCI inlet (AP probe) with the corona needle removed. Ionization was performed through selective resonance-enhanced multi-photon ionization schemes using a high-repetition-rate fixed-frequency excimer laser operating at 248 nm. Detection limits well within the low-fmol regime are readily obtained for various aromatic hydrocarbons that exhibit long-lived electronic states at the energy level of the first photon. Only molecular ions are generated at the low laser fluxes employed ( approximately 1 MW/cm(2)). The design and performance of the laser-ionization source are presented along with results of the analysis of aromatic hydrocarbons.

Water, treated as the continuous liquid in and around cells.

In the quantitative treatment of non-covalent inter- and intra-cellular interactions taking place in water, in vitro as well as in vivo, it is essential to treat the surrounding and pervading liquid medium as the continuous medium. In the close vicinity of inter- and intra-cellular surfaces and of biopolymers the various different non-covalent forces may locally alter the structure of water in a number of ways, but these local structural changes can be quantitatively taken into account. The operative forces are: Lifshitz-van der Waals (LW) forces. Lewis acid-base (AB) forces and electrostatic (EL) forces. Of these, the AB forces are generally the preponderant ones, in aqueous media. This is due, inter alia, to the strong cohesive and adhesive hydrogen-bonding interactions typically occurring in and by water. Among the strong AB interactions occurring in water are hydrophobic attraction (the hydrophobic effect) and hydrophilic repulsion (hydration pressure). Also treated is the function of LW, AB and EL forces in: hydration; in the stability of particle and cell suspensions, the solubility of biopolymers, small organic solutes, and electrolytes; and in specific ligand-receptor (such as antigen-antibody) interactions.

Retrospective clinical investigation of the impact of early treatment of children with Down's syndrome according to Castillo-Morales.

BACKGROUND: Infants and toddlers with Down's syndrome are treated at the Department of Orthodontics, University of Frankfurt/Main only when the tongue protrudes over the lower lip, hindering mouth closure. No plate therapy is applied in patients with less tongue protrusion. This study aimed to assess objectively the treatment effects of stimulation plate therapy after Castillo-Morales at this early stage of development. PATIENTS AND METHOD: The follow-up covered 33 children, 20 of whom showed no mouth closure with the tongue resting protrusively on the lower lip at first examination at the age of 8 months. These 20 children received orthodontic treatment based on a stimulation plate. The parents were advised to insert the plate four times a day for about half an hour respectively. The overall treatment time was ca. 2 years. The second group (13 children) received no early treatment, as the functional parameters were only slightly altered at the age of 7 months. At follow-up, the children of the treatment group were between 8.8 +/- 2.3, and those of the control group 8.9 +/- 3.0 years old. The children underwent clinical examination; the parents answered a questionnaire. Additionally, study casts and intraoral photographs were taken along with frontal and profile photographs. The factors assessed were various functions, dentition, facial development, and subjective rating of the parents. RESULTS: In contrast to the initial findings, no difference between the two groups was found at follow-up. CONCLUSION: Early treatment using a stimulation plate thus appears to mitigate or even normalize the initially more severe dysfunctions recorded in the study group as compared to the controls.

Electron-capture mass spectrometry: recent advances.

Electron-capture (EC) is a sensitive and selective ionization technique for mass spectrometry (MS). In the most familiar form of EC, a susceptible analyte (electrophore) is detected after eluting from a gas chromatography (GC) column, where a low attomole detection limit for standards is routine. High-performance liquid chromatography can facilitate sample cleanup prior to detection by GC-EC-MS, but carryover and shifts in retention time for the "invisible" analyte can be difficulties. Solid-phase extraction avoids these difficulties, but the degree of cleanup and recovery can be problems. Alternative electrophoric derivatizing reagents are available to help deal with interferences, and new reagents such as "AMACE1" are emerging. Releasable forms of electrophores can be used as tags for labeling macromolecules, motivated by the desire to multiplex ligand-type assays. The conventional, gas-phase ion source for EC is not well-understood, especially the role of wall reactions. Using an electron monochromator to tune the electron energy adds to the selectivity and information provided by EC-MS. High-resolution and tandem EC-MS measurements are emerging. Electron-capture dissociation is a new technique to sequence small- to medium-sized peptides, having the advantage of providing more extensive sequence information relative to other MS techniques. Particle-beam EC-MS tends to be less sensitive than GC-EC-MS, but not always. Recently it was demonstrated that EC-MS can be accomplished on an ordinary laser desorption time-of-flight mass spectrometer, and also by using atmospheric pressure chemical ionization. Two applications are discussed here in detail: bile acids and oxidized phenylalanine. EC-MS is well-established as a useful technique for trace analysis in special cases, and the scope of its usefulness is broadening (qualitative analysis and detection of more polar and larger molecules), based on advances in both the chemical and instrumental aspects of this technique.

AMACE1: versatile aminoacetamide electrophore reagent.

Acetamide, 2-amino-N-[[3,5-bis(trifluoromethyl)phenyl]-methyl]-N-methyl-, monohydrochloride, which we have named AMACE1, was synthesized in three steps starting from N-tritylglycine. AMACE1 was coupled via its primary amine group (pKa 8.2) under aqueous conditions to four model analytes for oxidative sugar damage to DNA: glycolate, 3-hydroxy-2-butanone, 3-phenylbutyraldehyde, and alpha-hydroxy-gamma-butyrolactone, relying on cyanoborohydride for coupling to a keto function and a water-soluble carbodiimide for coupling to a carboxyl function. Further reaction with butyric anhydride led to products that could be detected by gas chromatography/electron capture mass spectrometry when 1 microL of ethyl acetate containing essentially 20 amol of each product was injected, on the basis of selected ion monitoring of the analyte characteristic anion fragment from dissociative loss of the 3,5-bis-(trifluoromethyl)phenylmethyl moiety: m/z 215, 289, 299, and 329, respectively. Since many small, organic analytes contain a keto or carboxylic acid group, AMACE1 should be useful in general in the area of trace organic analysis.

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry method for analysis of endogenous and exogenous N7-(2-hydroxyethyl)guanine in rodents and its potential for human biological monitoring.

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS) method was developed for quantitating N7-(2-hydroxyethyl)guanine (N7-HEG) with excellent sensitivity and specificity. [4,5,6,8-(13)C(4)]-N7-HEG was synthesized, characterized, and quantitated using HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) so it could serve as an internal standard. After being converted to its corresponding xanthine and derivatized with pentafluorobenzyl (PFB) bromide twice, the PFB derivative of N7-HEG was characterized using GC/EC/NCI-HRMS carried out at full scan mode. The most abundant fragment was at m/z 555, with a molecular formula of C(21)H(9)N(4)O(3)F(10), resulting from the loss of one PFB group. By monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard), this assay demonstrated a linear relationship over a range of 1 fmol to 1 pmol of N7-HEG versus 20 fmol of [(13)C(4)]-N7-HEG on column. The limit of detection (LOD) for the complete assay was 600 amol (S/N = 5) injected on column. The variation of this assay was within 15% from 1 to 20 fmol of N7-HEG versus 2 fmol of [(13)C(4)]-N7-HEG with four replications for each calibration standard. Two hundred to three hundred micrograms of spleen DNA of control rats and mice and 100 microg of spleen DNA of rats and mice exposed to 3000 ppm ethylene for 6 h/day for 5 days were analyzed using GC/EC/NCI-HRMS. The amounts of N7-HEG varied from 0.2 to 0.3 pmol/micromol of guanine in tissues of control rats. Ethylene-exposed animals had 5-15-fold higher N7-HEG levels than controls. This assay was able to quantitate N7-HEG in 25-30 microg of DNA from human lymphocytes with excellent specificity. This was due in part to human tissues having 10-15-fold higher amounts of endogenous N7-HEG than rodents. These results show that this GC/EC/NCI-HRMS method is highly sensitive and specific and can be used in biological monitoring and molecular dosimetry and molecular epidemiology studies.

Matrix-assisted laser desorption/ionization mass spectrometry of deoxynucleotides labeled with an IMI dye.

The four major deoxynucleotides of DNA, and adduct mixtures resulting from separate reactions of 5'-dAMP and 5'-dGMP with benzo[a]pyrene diolepoxide (BPDE), were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) after labeling of their phosphate group with an IMI dye. The latter reagent comprises an imidazole functional group attached to a BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene+ ++) fluorophore. Good sensitivity was observed in the detection of the IMI-labeled products by MALDI-MS: 300-500 fmol in the laser spot (1% of the 30-50 pmol sample on the target) gave a signal-to-noise (S/N) of >/=30 from 20-30 superimposed laser shots. The BPDE reaction products, after the IMI labeling, were also subjected to capillary electrophoresis with laser-induced fluorescence detection, which revealed a complex mixture of products. Overall the results encourage the further development of this 'IMI-postlabeling' methodology as an alternative to (32)P-postlabeling for the detection of DNA adducts.

Preparation of an IMI dye (imidazole functional group) containing a 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole fluorophore for labeling of phosphomonoesters.

We are studying dye-imidazole conjugates ("IMI dyes") as reagents for labeling phosphomonoesters such as nucleotides. Previously we have employed a BODIPY dye in our IMI reagents, and analyzed the labeled products by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) involving an argon ion laser. (The BODIPY fluorophore is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Here we broaden the technology by preparing a DBD-IMI dye [DBD = 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole], and using a helium-cadmium laser. While DBD-IMI (IMI3) is about 50x more stable photolytically than a BODIPY-IMI dye (IMI2, a conjugate of a BODIPY dye with histamine, was tested), the detection limit for IMI2 (5.10(-11) M; S/N = 5, CE-LIF with an argon ion laser) is tenfold better than that for IMI3 (5.10(-10) M, S/N = 5, helium-cadmium laser). IMI3 conjugates of the four major DNA nucleotides were prepared and detected by CE-LIF.

Contrasting behavior of pentafluorophenoxyacetone and pentafluorobenzyloxyacetone in electron impact and electron capture mass spectrometry.

Towards the goal of finding new ketone electrophores suitable as molecular labels for electrophoric release tags, pentafluorophenoxyacetone (1) and pentafluorobenzyloxyacetone (2) were prepared. Both ketones were evaluated by electron capture (EC) and electron impact (EI) modes of mass spectrometry (MS). By EC-MS, 1 nearly gave a single ion (as desired), whereas 2 gave many ions. This behavior was completely reversed in EI-MS. To account for certain ion fragments in the EC mass spectrum of 2, an anion radical McLafferty-type rearrangement and loss of a carbene neutral were postulated. Electron impact of 1 gave an abundant ion at m/z 117 (C5F3+), which was suggested to be a diyne cation.

Phosphate-specific fluorescence labeling with BO-IMI: reaction details.

Previously we reported than BO-IMI, a reagent which contains a BODIPY fluorophore linked to an imidazole group, can be used to covalently label a phosphomonoester in a single step under aqueous conditions [P. Wang, R.W. Giese, Anal. Chem. 65 (1993) 3518]. The reaction was conducted in the presence of a water-soluble carbodiimide 1-ethyl-3-(3'-N,N'-dimethylaminopropyl) carbodiimide [EDC] to activate the phosphomonoester, and the coupling took place onto both the N1 and N3 imidazole nitrogens of BO-IMI. Whether the two BO-IMI-phosphomonoester regioisomers migrated separately or together during capillary electrophoresis depended on the pH, due to a difference in their pKa values. Since then, we have studied the reaction in more detail leading to the information reported here. First, we have learned that the regioisomer ratio changes during the course of the reaction, and found that the mechanism involves both spontaneous and BO-IMI-catalyzed hydrolysis of the less stable isomer. Second, there is a background reaction in which BO-IMI becomes attached to EDC. Third, the BO-IMI-phosphomonoester product (a mixture of two isomers), that is observed by capillary electrophoresis at an alkaline pH, is found to no longer contain the two fluorine atoms present in the starting BO-IMI reagent. This is because they are placed by hydroxy groups at high pH. Finally, an event was discovered which complicates the detection of less than about 60 fmol of a phosphomonoester with BO-IMI: hydrolysis of a tiny fraction of the BO-IMI takes place during the coupling reaction, which leads to chemical noise in the capillary electropherogram.

N-hydroxysuccinimide ester labeling 5'-aminoalkyl DNA oligomers: reaction conditions and purification.

Difficulties were encountered in labeling 5'-aminoalkyl DNA oligomers with glycolketo electrophore N-hydroxysuccinimide esters in aqueous sodium bicarbonate (a common base for this purpose), followed by C18-silica reversed-phase high-performance liquid chromatography (HPLC) to achieve purification. The electrophore-labeled oligomers were not separated readily either from the hydrolyzed electrophore or from the starting oligomer. This problem was overcome by conducting the reaction with triethylamine as a base, organic washing the reaction mixtures after evaporation, and separating on a C18-poly(styrene-divinylbenzene) HPLC packing.

Electrophore mass tag dideoxy DNA sequencing.

Toward a goal of dideoxy sequencing DNA utilizing electrophore labels, we prepared four electrophore-labeled DNA oligonucleotide primers. Each primer has a different electrophore and DNA sequence but a common glycol keto (alpha,beta-dihydroxyketo) release group. Cleavage of this latter group by either periodate oxidation or a thermal retroaldol reaction releases the electrophores for detection by mass spectrometry. Successful sequencing data with these primers was obtained by capillary electrophoresis on an ABI Model 310 after fluorescence dideoxy terminator cycle sequencing reactions were conducted. In a separate experiment, it was demonstrated that a cocktail of the four electrophore DNA primers could be detected as a dried sample spot by CO2 laser desorption/capillary collection/gas chromatography electron capture mass spectrometry. These results establish some feasibility for our long-term goal of high-speed multiplex electrophore mass tag dideoxy DNA sequencing. Ultimately we plan to use a higher number of electrophore mass tags and to rely on direct detection of the desorbed electrophores by electron capture time-of-flight mass spectrometry.