Training of attention functions in children with attention deficit hyperactivity disorder

Pharmacological treatment of children with ADHD has been shown to be successful; however, medication may not normalize attention functions. The present study was based on a neuropsychological model of attention and assessed the effect of an attention training program on attentional functioning of children with ADHD. Thirty-two children with ADHD and 16 healthy children participated in the study. Children with ADHD were randomly assigned to one of the two conditions, i.e., an attention training program which trained aspects of vigilance, selective attention and divided attention, or a visual perception training which trained perceptual skills, such as perception of figure and ground, form constancy and position in space. The training programs were applied in individual sessions, twice a week, for a period of four consecutive weeks. Healthy children did not receive any training. Alertness, vigilance, selective attention, divided attention, and flexibility were examined prior to and following the interventions. Children with ADHD were assessed and trained while on ADHD medications. Data analysis revealed that the attention training used in the present study led to significant improvements of various aspects of attention, including vigilance, divided attention, and flexibility, while the visual perception training had no specific effects. The findings indicate that attention training programs have the potential to facilitate attentional functioning in children with ADHD treated with ADHD drugs

DNA-Bindung und Modifizierung der DNA-bindenden Domäne des bakteriellen Transkriptionsfaktors PhoB aus E. coli

Niemann G. DNA-Bindung und Modifizierung der DNA-bindenden Domäne des bakteriellen Transkriptionsfaktors PhoB aus E. coli. Bielefeld: Universität Bielefeld; 2014

Minor Groove Recognition is Important for the Transcription Factor PhoB: A Surface Plasmon Resonance Study

Ritzefeld M, Wollschläger K, Niemann G, Anselmetti D, Sewald N. Minor Groove Recognition is Important for the Transcription Factor PhoB: A Surface Plasmon Resonance Study. Molecular Biosystems. 2011;7(11):3132-3142.The two-component regulatory system PhoR/PhoB induces the expression of several genes in response to phosphate starvation in Escherichia coli. In order to quantify these protein–DNA interactions and to study the time-resolved dynamics of the binding mechanism, the specific recognition of different oligonucleotide duplexes by the DNA-binding domain of PhoB (PhoBDBD) was analyzed using surface plasmon resonance. In addition the two point mutants PhoBDBDD196A and PhoBDBDR219A were obtained and the DNA recognition in comparison to the wildtype PhoBDBD was investigated. Aspartic acid 196 and arginine 219 mediate specific minor groove interactions. All results reveal that at high PhoBDBD-concentrations all recognition sequences of the pho box are occupied. Decreasing the protein amount results in a mixture of free oligonucleotides and DNA molecules occupied by two WT-PhoBDBD. Moreover, the SPR results indicate that both binding site segments, the TGTCA-motif and the A/T-rich minor groove, are essential for the binding process. A comparison of different regulons additionally proved the dependency of the recognition process on the base composition of the minor groove

In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations

Gaertner A, Klauke B, Stork I, Niehaus K, Niemann G. In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations. PloS one. 2012;7(10): e47097.BACKGROUND: Although numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate. METHODOLOGY/PRINCIPAL FINDINGS: To test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation. CONCLUSIONS/SIGNIFICANCE: The ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study

Adhesion properties of rECDs.

<p><b>A+B</b> Flow cytometry-based assay for the binding of 0.8 µM rECD-wt or -variants to HT1080. <b>A</b> Representative histograms of FITC- fluorescence for binding of rECD-wt- and rECD-R46Q (as indicated). Bound rECD was detected with anti-HisFITC. As a negative control, HT1080 cells were incubated with only anti-HisFITC (grey filled area). <b>B</b> Column plots representing the ratio of rECD-binding related to the negative control (ratio_rECD-bound) as detected by flow cytometry. Ratios_rECD-bound are indicated as mean± SEM of 7 independent measurements for rECD-variants and 9 independent measurements for rECD-wt with rECDs from at least 3 different purifications. Statistical analysis was performed by one-way ANOVA with Dunnett’s posttest using rECD-wt as a control (GraphPad Prism 5.01). rECD-R46Q-binding to HT1080 is increased 1.8-fold as compared to rECD-wt. Other ARVC-associated variants have no influence on rECD-binding to HT1080. <b>C</b> Representative Western blot (with anti-DSG2-10G11) of rECDs crosslinked in a 5 mM CaCl₂ containing buffer with BS³ (+) or of controls (-) reveals that rECD wild-type and variants exist in solution as monomers (m), dimers (d), and oligomers (o).</p

Representative images for detecting the localisation of wild-type and variants of full-length-DSG2-EYFP in HT1080 for three independent transfection experiments:

<p><b>DSC2b-HT1080 cells were transfected with full-length(fl)-DSG2-pEYFP; live cells were analysed with a fluorescence miscroscope one day after transfection.</b> R46Q, D154E, D187G, K294E and V392I indicate the sequence variant in fl-DSG2-EYFP, wt fl-DSG2-wt-pEYFP, and C the LFA mock transfected control. Chimeric DSG2-proteins localised preferentially to the cell borders. ARVC-associated variations had no detectable influence on the localisation of fl-DSG2-EYFP in DSC2b-HT1080. Images were acquired through YFP and phase-contrast filters. Scale (red bar) = 10 µm.</p

Evaluation of CD data.

<p>Results of the deconvolution with DichroWeb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Whitmore1" target="_blank">[67]</a> using the CONTINLL-algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Provencher1" target="_blank">[68]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanStokkum1" target="_blank">[69]</a> and the CRYST175 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Evans1" target="_blank">[70]</a> reference data set. The results are presented as means±SEM [%] for three independent measurements. α-helical content (<b>A</b>) was 5.9±0.8 and 5.9±0.5 without Ca²⁺ (-Ca²⁺) and 3.2±1.4 and 3.2±0.2 with 5 mM CaCl₂ for rECD-wt-nat and rECD-wt-denat, respectively. β-strand content (<b>B</b>) was 38.2±0.3 and 36.6±1.3 without Ca²⁺ and 40.5±0.8 and 40.7±0.6 with 5 mM CaCl₂. Analysis of the secondary structure with two-way ANOVA showed that the α-helix content (<b>A</b>) was significantly decreased (p<0.05) while the β-strand content (<b>B</b>) was significantly increased (p<0.01) by the addition of 5 mM CaCl₂ (+Ca²⁺). However, as shown by two-way ANOVA, purification conditions had no significant effect on the rECD secondary structure.</p

Investigated DSG2-variants.

<p>All data were obtained from the ARVC database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanderZwaag1" target="_blank">[64]</a> and the corresponding references. TFC = task force criteria, EC = extracellular cadherin domain, DCM = dilatative cardiomyopathy.</p>a<p>The prevalence in controls for the DSG2-V392I was adapted to the results in our research group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Klauke1" target="_blank">[35]</a>.</p>b<p>Grantham, R. (1974). “Amino acid difference formula to help explain protein evolution.” Science 185(4154):862–864.; Li, W. H., C. I. Wu, et al. (1984). “Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications.” J Mol Evol 21(1): 58–71.</p>c<p>Kumar, P., S. Henikoff, et al. (2009). “Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.” Nat Protoc 4(7): 1073–1081.</p>d<p>Ramensky, V., P. Bork, et al. (2002). “Human non-synonymous SNPs: server and survey.” Nucleic Acids Res 30(17): 3894–3900.</p

Comparison of rECD fragment ion peaks generated by MALDI-ISD using QuPE [<b>71</b>].

<p><b>A</b> The location of <i>ECD</i> and <i>Pro-ECD</i> and the positions of the corresponding c-ions are illustrated on the schematic view of the rECD. <b>B+C</b> Each row shows extracts of the MALDI-ISD spectra for the particular rECDs. Peaks in one column represent the ions of the m/z ratios indicated below the negative control. The corresponding c-ions are indicated for each peak. The calculated monoisotopic masses [M+H]⁺ are shown at the bottom of each column. <b>B</b> Fragment ion peaks representing the <i>ECD</i>-c-ions; only rECD-R46Q shows no fragment ions corresponding to <i>ECD </i><b>C</b> Fragment ion peaks representing the <i>Pro-ECD-</i>c-ions; only rECD-R46Q shows fragment ions corresponding to <i>Pro-ECD</i>.</p

Flow cytometry-based assay for rECD-binding.

<p><b>A</b> Representative histograms of FITC-fluorescence for rECD-wt binding with 5 mM CaCl₂ (1) or with 2 mM EGTA (2). HT1080 cells were incubated with (black line) or without (negative control, grey filled area) rECD-wt. Bound rECD-wt was detected with anti-HisFITC. <b>B</b> Column plots representing the ratios of rECD-wt-binding (ratio_rECD-bound; for calculation see Supporting Information) indicated as mean±SEM of 3 independent measurements as detected by flow cytometry. The ratio_rECD-bound was significantly (p<0.01) decreased from 1.21±0.03 for samples incubated in 5 mM CaCl₂ (1) to 1.05±0.03 for samples incubated with 2 mM EGTA (2) showing that rECD-wt has a Ca²⁺-dependent binding to HT1080 cells. Statistical analysis was performed with unpaired student’s t-test (GraphPad Prism 5.01).</p