Cytokine levels in children and adults with wheezing and asthma show specific patterns of variability over time.

Levels of cytokines are used for in-depth characterization of patients with asthma; however, the variability over time might be a critical confounder. To analyze the course of serum cytokines in children, adolescents and adults with asthma and in healthy controls and to propose statistical methods to control for seasonal effects. Of 532 screened subjects, 514 (91·5%) were included in the All Age Asthma Cohort (ALLIANCE). The cohort included 279 children with either recurrent wheezing bronchitis (more than two episodes) or doctor-diagnosed asthma, 75 healthy controls, 150 adult asthmatics and 31 adult healthy controls. Blood samples were collected and 25 µl serum was used for analysis with the Bio-Plex Pr human cytokine 27-Plex assay. Mean age, body mass index and gender in the three groups of wheezers, asthmatic children and adult asthmatics were comparable to healthy controls. Wheezers (34·5%), asthmatic children (78·7%) and adult asthmatics (62·8%) were significantly more often sensitized compared to controls (4·5, 22 and 22·6%, respectively). Considering the entire cohort, interleukin (IL)-1ra, IL-4, IL-9, IL-17, macrophage inflammatory protein (MIP)-1- α and tumor necrosis factor (TNF)- α showed seasonal variability, whereas IL-1ß, IL-7, IL-8, IL-13, eotaxin, granulocyte colony-stimulating factor (G-CSF), interferon gamma-induced protein (IP)-10, MIP-1 ß and platelet-derived growth factor (PDGF)-BB did not. Significant differences between wheezers/asthmatics and healthy controls were observed for IL-17 and PDGF-BB, which remained stable after adjustment for the seasonality of IL-17. Seasonality has a significant impact on serum cytokine levels in patients with asthma. Because endotyping has achieved clinical importance to guide individualized patient-tailored therapy, it is important to account for seasonal effects.

Probing hydrogen bonding orbitals: resonant inelastic soft X-ray scattering of aqueous NH3.

To probe the influence of hydrogen bonding on the electronic structure of ammonia, gas phase and aqueous NH3 have been investigated using soft X-ray absorption (XAS), resonant inelastic soft X-ray scattering (RIXS), and electronic structure calculations including dynamical effects. Strong spectral differences in the XAS scans as well as in the RIXS spectra between gas phase and aqueous NH3 are attributed to orbital mixing with the water orbitals, dipole-dipole interactions, differences in vibronic coupling, and nuclear dynamics on the time-scale of the RIXS process. All of these effects are consequences of hydrogen bonding and the impact of the associated orbitals, demonstrating the power of XAS and RIXS as unique tools to study hydrogen bonding in liquids.

The Be K-edge in beryllium oxide and chalcogenides: soft x-ray absorption spectra from first-principles theory and experiment.

We have carried out a theoretical and experimental investigation of the beryllium K-edge soft x-ray absorption fine structure of beryllium compounds in the oxygen group, considering BeO, BeS, BeSe, and BeTe. Theoretical spectra are obtained ab initio, through many-body perturbation theory, by solving the Bethe-Salpeter equation (BSE), and by supercell calculations using the core-hole approximation. All calculations are performed with the full-potential linearized augmented plane-wave method. It is found that the two different theoretical approaches produce a similar fine structure, in good agreement with the experimental data. Using the BSE results, we interpret the spectra, distinguishing between bound core-excitons and higher energy excitations.

Ultrafast proton dynamics in aqueous amino acid solutions studied by resonant inelastic soft X-ray scattering.

Resonant inelastic soft X-ray scattering (RIXS) has been used to study the electronic structure of glycine and lysine in aqueous solution. Upon variation of the pH value of the solution from acidic to basic, major changes of the nitrogen K edge RIXS data are observed for both amino acids, which are associated with the protonation and deprotonation of the amino groups. The experimental results are compared with simulations based on density functional theory, yielding a detailed understanding of the spectral changes, as well as insights into the ultrafast proton dynamics in the intermediate core-excited/ionized state of the RIXS process.

Real-time PCR quantification of major Wilms' tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences.

Wilms' tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5-/KTS-], B[+/-], C[-/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features-overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[-] ratio was less variable than the EX5[+]/EX5[-] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[-] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.

Electron-hole correlation effects in core-level spectroscopy probed by the resonant inelastic soft x-ray scattering map of C60.

We have employed a unique spectroscopic approach, a resonant inelastic soft x-ray scattering (RIXS) map, to identify and separate electron-hole correlation effects in core-level spectroscopy. With this approach, we are able to derive a comprehensive picture of the electronic structure, separating ground state properties (such as the HOMO-LUMO separation) from excited state properties (such as the C 1s core-exciton binding energy of C(60)). In particular, our approach allows us to determine the difference between core- and valence exciton binding energies in C(60) [0.5 (±0.2) eV]. Furthermore, the RIXS map gives detailed insight into the symmetries of the intermediate and final states of the RIXS process.

Normative data for lung function and exhaled nitric oxide in unsedated healthy infants.

Despite association with lung growth and long-term respiratory morbidity, there is a lack of normative lung function data for unsedated infants conforming to latest European Respiratory Society/American Thoracic Society standards. Lung function was measured using an ultrasonic flow meter in 342 unsedated, healthy, term-born infants at a mean ± sd age of 5.1 ± 0.8 weeks during natural sleep according to the latest standards. Tidal breathing flow-volume loops (TBFVL) and exhaled nitric oxide (eNO) measurements were obtained from 100 regular breaths. We aimed for three acceptable measurements for multiple-breath washout and 5-10 acceptable interruption resistance (R(int)) measurements. Acceptable measurements were obtained in ≤ 285 infants with high variability. Mean values were 7.48 mL·kg⁻¹ (95% limits of agreement 4.95-10.0 mL·kg⁻¹) for tidal volume, 14.3 ppb (2.6-26.1 ppb) for eNO, 23.9 mL·kg⁻¹ (16.0-31.8 mL·kg⁻¹) for functional residual capacity, 6.75 (5.63-7.87) for lung clearance index and 3.78 kPa·s·L⁻¹ (1.14-6.42 kPa·s·L⁻¹) for R(int). In males, TBFVL outcomes were associated with anthropometric parameters and in females, with maternal smoking during pregnancy, maternal asthma and Caesarean section. This large normative data set in unsedated infants offers reference values for future research and particularly for studies where sedation may put infants at risk. Furthermore, it highlights the impact of maternal and environmental risk factors on neonatal lung function.

A functional magnetic resonance imaging navigated repetitive transcranial magnetic stimulation study of the posterior parietal cortex in normal pain and hyperalgesia.

Noxious stimuli activate a complex cerebral network. During central sensitization to pain, activity in most of these areas is changed. One of these areas is the posterior parietal cortex (PPC). The role of the PPC during processing of acute pain as well as hyperalgesia and tactile allodynia remains elusive. Therefore, we performed a functional magnetic resonance imaging (fMRI) based, neuro-navigated, repetitive transcranial magnetic stimulation (rTMS) study in 10 healthy volunteers. Firstly, pin-prick hyperalgesia was provoked on the right volar forearm, using the model of electrically-induced secondary mechanical hyperalgesia. fMRI was performed during pin-prick stimulation inside and outside the hyperalgesic areas. Secondly, on four different experimental sessions, the left and right individual intraparietal BOLD peak-activations were used as targets for a sham-controlled 1 Hz rTMS paradigm of 10 min duration. We measured psychophysically the (i) electrical pain stimulus intensity on an 11-point numeric pain rating scale (NRS, 0-10), the (ii) area of hyperalgesia, and the (iii) area of dynamic mechanical allodynia. Sham stimulation or rTMS was performed 16 min after induction of pin-prick hyperalgesia and tactile allodynia. Compared to sham stimulation, no significant effect of rTMS was observed on pain stimulus intensity and the area of allodynia. However, a reduction of the hyperalgesic area was observed for rTMS of the left PPC (P<0.05). We discuss the role of the PPC in central sensitization to pain, in spatial discrimination of pain stimuli and in spatial-attention to pain stimuli.

CCAAT/enhancer-binding protein alpha (CEBPA) polymorphisms and mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia.

The CCAAT/enhancer-binding protein alpha, encoded by the intronless CEBPA gene, is a transcription factor that induces expression of genes involved in differentiation of granulocytes, monocytes, adipocytes and hepatocytes. Both mono- and bi-allelic CEBPA mutations were detected in acute myeloid leukaemia and myelodysplastic syndrome. In this study we also identified CEBPA mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia. We found 16 various deletions with the presence of two direct repeats in CEBPA by analysis of 431 individuals. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493- 498_865-870), GG (486-487_885-886), and GCCAAGCAGC (508-517_907-916), all according to GenBank Accession No. NM_004364.2. In one case we identified that a father with ischaemic heart disease and his healthy son had two identical deletions (493_864del and 508_906del, both according to GenBank Accession No. NM_004364.2) in CEBPA. The occurrence of deletions between two repetitive sequences may be caused by recombination events in the repair process. A double-stranded cut in DNA may initiate these recombination events in adjacent DNA sequences. Four types of polymorphisms in the CEBPA gene were also detected in the screened individuals. Polymorphism in CEBPA gene 690 G>T according to GenBank Accession No. NM_004364.2 is the most frequent type in our analysis. Statistical analysis did not find significant differences in the frequency of polymorphisms in CEBPA in patients and in healthy individuals with the exception of P4 polymorphism (580_585dup according to GenBank Accesion No. NM_004364.2). P4 polymorphism was significantly increased in ischaemic heart disease patients.

High-resolution, high-transmission soft x-ray spectrometer for the study of biological samples.

We present a variable line-space grating spectrometer for soft x-rays that covers the photon energy range between 130 and 650 eV. The optical design is based on the Hettrick-Underwood principle and tailored to synchrotron-based studies of radiation-sensitive biological samples. The spectrometer is able to record the entire spectral range in one shot, i.e., without any mechanical motion, at a resolving power of 1200 or better. Despite its slitless design, such a resolving power can be achieved for a source spot as large as (30 x 3000) microm2, which is important for keeping beam damage effects in radiation-sensitive samples low. The high spectrometer efficiency allows recording of comprehensive two-dimensional resonant inelastic soft x-ray scattering (RIXS) maps with good statistics within several minutes. This is exemplarily demonstrated for a RIXS map of highly oriented pyrolytic graphite, which was taken within 10 min.

Solid and liquid spectroscopic analysis (SALSA)--a soft x-ray spectroscopy endstation with a novel flow-through liquid cell.

We present a novel synchrotron endstation with a flow-through liquid cell designed to study the electronic structure of liquids using soft x-ray spectroscopies. In this cell, the liquid under study is separated from the vacuum by a thin window membrane, such that the sample liquid can be investigated at ambient pressure. The temperature of the probing volume can be varied in a broad range and with a fast temperature response. The optimized design of the cell significantly reduces the amount of required sample liquid and allows the use of different window membrane types necessary to cover a broad energy range. The liquid cell is integrated into the solid and liquid spectroscopic analysis (SALSA) endstation that includes a high-resolution, high-transmission x-ray spectrometer and a state-of-the-art electron analyzer. The modular design of SALSA also allows the measurement of solid-state samples. The capabilities of the liquid cell and the x-ray spectrometer are demonstrated using a resonant inelastic x-ray scattering map of a 25 wt % NaOD solution.

Isotope and temperature effects in liquid water probed by x-ray absorption and resonant x-ray emission spectroscopy.

High-resolution x-ray absorption and emission spectra of liquid water exhibit a strong isotope effect. Further, the emission spectra show a splitting of the 1b1 emission line, a weak temperature effect, and a pronounced excitation-energy dependence. They can be described as a superposition of two independent contributions. By comparing with gas phase, ice, and NaOH/NaOD, we propose that the two components are governed by the initial state hydrogen bonding configuration and ultrafast dissociation on the time scale of the O 1s core hole decay.

Congenital hypothyroidism caused by a novel homozygous mutation in the thyroid peroxidase gene.

UNLABELLED: Congenital primary hypothyroidism occurs in one out of 4,000 births. About 20% of cases are due to defects in thyroid hormonogenesis. We report on a German girl with congenital hypothyroidism due to a mutation in the thyroid peroxidase (TPO) gene who had elevated serum levels of thyroglobulin during periods of hyperthyrotropinemia. METHODS: The TPO gene was sequenced directly from genomic DNA. RESULTS: The patient had a novel homozygous mutation (R314W) in the TPO gene. The unaffected parents were non-consanguineous and both heterozygous carriers of the mutation. Fifty normal individuals did not harbor the mutation ruling out a common polymorphism. CONCLUSION: The identified TPO gene mutation (R314W) is very likely the genetic cause for hypothyroidism in the reported child. R314W has not been described before and codes for a presumably inactive TPO molecule.

Growth-inhibiting activity of transcription factor C/EBPalpha, its role in haematopoiesis and its tumour suppressor or oncogenic properties in leukaemias.

The CCAAT/enhancer binding protein alpha (C/EBPalpha or CEBPA) is the founding member of a family of related leucine zipper transcription factors that play important roles in myeloid differentiation. Targeted inactivation of C/EBPalpha in mice demonstrates its importance in the proper development and function of liver, adipose tissue, lung and haematopoietic tissues. C/EBPalpha is highly expressed in these differentiated tissues where it controls differentiation-dependent gene expression and inhibits cell proliferation. Learning more about the precise molecular functions of the C/EBPalpha protein and how these are affected by leukaemogenic mutations should lead to an improved understanding of the cellular functions that are disrupted in patients with AML. Decreased expression of C/EBPalpha but not C/EBPalpha mutation has been shown in patients with granulocytic leukaemias that are associated with translocations t(8;21), inv (16) or t(15;17). Derived fusion proteins repress C/EBPalpha expression. Differentiation therapy of some AML types is based on restoring C/EBPalpha function. However, apparently normal C/EBPalpha is overexpressed in BCP-ALL harbouring the translocation t(14; 19)(q32; q13). C/EBPalpha may exhibit oncogenic as well as tumour suppressor properties in human leukaemogenesis. C/EBPalpha mutations were not found in non-haematopoietic cancers. DNA hypermethylation of the upstream C/EBPalpha promoter region is responsible for very low C/EBPalpha expression in human lung and endometrial cancer. C/EBPalpha expression may be a biomarker for early detection of these cancers and DNA-modifying drugs such as demethylating agents and/or histone deacetylase inhibitors could be used in the treatment of these malignancies.

[EVI1 and its role in myelodysplastic syndrome, myeloid leukemia and other malignant diseases]. / EVI1 a jeho úloha u myelodysplastických syndromu, myeloidní leukémie a dalsích maligních onemocnení.

The ecotropic viral integration site 1 (EVI1) gene was identified as a common locus of retroviral integration in myeloid tumors found in mice. EVI1 gene is highly conserved through evolution and human gene EVI1 on chromosome 3q26 encodes zinc fingers-containing transcription factor. EVI1 is expressed in nonhematopoietic tissues but not in normal blood or bone marrow. EVI1 was detected in hematopoietic cells in retrovirus-induced myeloid leukemias in mice and several reports documented EVI1 expression in human myelodysplastic syndromes and other hematologic malignancies without 3q26 translocations. EVI1 is abnormally expressed in human myeloid leukemias that are associated with the t(3;3)(q21;q26), t(3;21)(q26;q22), inv(3)(q21q26) and other chromosomal rearrangements. EVI1 is overexpressed in some ovarian cancers and human colon cancer cell lines and may play a role in the initiation and/or progression of solid tumors, as well as hematopoietic malignancies. EVI1 is a transcriptional repressor which inhibits transforming growth factor beta (TGFbeta) family signalling by binding signal transducers (Smad proteins) and recruiting transcriptional corepressors. TGFbeta is an important regulator of proliferation, differentiation, apoptosis and migration of cells. EVI1 inhibits TGFbeta-mediated apoptosis. Knockdown of EVI1 function by small interference RNA increases the sensitivity of malignant cells to TGFbeta-mediated or other inducer-mediated apoptosis. Overexpressed EVI-1 blocks granulocyte and erythroid differentiation and possess the ability of growth promotion in some types of cells. EVI1 functions in some cases as a transcriptional activator which stimulates for example GATA2 and GATA3 promoters. The study of EVI1 target genes will help to clear the mechanism by which EVI1 upregulates cell proliferation, impairs cell differentiation, and induces cell transformation.

[Ubiquitins, proteasomes, sumoylation and application today and in future for cancer and other diseases therapy II. Sumoylation and neddylation as posttranslational modifications of proteins and their ubiquitinylation and its significance]. / Ubikvitiny, proteazomy, sumoylace a pouzití dnes a zítra v terapli nádoru I jiných chorob II. Sumoylace a neddylace jako posttranslacní modifikace proteinu podobné ubikvitinylaci a jelich význam.

Posttranslational protein modifications are effective devices that cells use to control the functions of proteins. Ubiquitin-like protein modifiers (Ubls) are posttranslationally attached to proteins by enzymatic reactions that are similar to ubiquitin conjugation. SUMO (small ubiquitin-related modifier) family proteins are the most intriguing Ubls. Sumoylation is the covalent attachment of SUMO to target proteins. Neddylation is the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins. Cullin family proteins organize ubiquitin ligase complexes to target numerous cellular proteins for polyubiquitinylation and subsequent proteasomal degradation. Despite the similarities in their structure and in enzymatic reactions Ubls and ubiquitin have distinct functions. In contrast with polyubiquitinylation that targets modified proteins to proteasome degradation, the biological consequences of sumoylation include the increase of protein stability. Sumoylation also helps in the protein transport from the cytoplasm to nucleus of cells, regulates transcriptional activities of proteins and mediates the binding of the protein to other proteins. Neddylation has importance for cell cycle control, signal transmission, cell differentiation and DNA repair. Recent studies linked sumoylation and neddylation of several proteins to important diseases (neurodegenerative diseases, acute promyelocytic leukemia, type I diabetes and other disorders). The regulation of these postranslational modifications may provide new targets for therapeutic intervention in several human diseases.

[Ubiquitins, proteasomes, sumoylation and therapeutic application today and in future for cancer and other diseases. I. Ubiquitin-proteasome system and the transcription factor NF-kappaB]. / Ubikvitiny, proteazomy, sumoylace a pouzití dnes a zítra v terapii nádoru i jiných chorob I. Ubikvitin-proteazomový system a transkripcní faktor NF-kappaB.

Proteasome is protein complex with proteolytic activity. Proteasomes are in addition to lysosomes the main proteolytic machinery of the eukaryotic cell. Proteins destined for degradation in proteasomes are marked by ubiquitinylation, which consists in attachment of polyubiquitin to relevant protein. The transport of polyubiquitinylated protein follows to proteasome, where protein is cleaved into small peptides. Besides polyubiquitin attachment to protein, monoubiquitinylation of proteins exists and has an important role in DNA repair, transcription of genes, endocytosis and signal transduction. The function of an important transcription factor NF-kappaB is connected with proteasome. NF-kappaB is activated after the proteolysis of its inhibitor IkappaB in proteasome. Ubiqutinylation and degradation of protein in proteasome and the activation of NF-kappaB play significant roles in taking proteins away and in expression of great numbers of genes important for the regulation of the cell cycle and apoptosis of cells. The inhibition of proteasomes has antiproliferative and antiinflammatory effects and opens new therapeutic approaches to a treatment of cancer and some inflammatory diseases. We divided the review into three parts: I. Ubiquitin-proteasome system and the transcription factor NF-kappaB, II. Sumoylation and neddylation as post-translational modification of proteins similar to ubiquitinylation and their significance and lastly III. Using of the knowledge of ubiquitin-proteasome system in cancer and other diseases therapy.

Spectroscopic investigation of the deeply buried Cu(In,Ga)(S,Se)2/Mo interface in thin-film solar cells.

The Cu(In,Ga)(S,Se)(2)Mo interface in thin-film solar cells has been investigated by surface-sensitive photoelectron spectroscopy, bulk-sensitive x-ray emission spectroscopy, and atomic force microscopy. It is possible to access this deeply buried interface by using a suitable lift-off technique, which allows us to investigate the back side of the absorber layer as well as the front side of the Mo back contact. We find a layer of Mo(S,Se)(2) on the surface of the Mo back contact and a copper-poor stoichiometry at the back side of the Cu(In,Ga)(S,Se)(2) absorber. Furthermore, we observe that the Na content at the Cu(In,Ga)(S,Se)(2)Mo interface as well as at the inner grain boundaries in the back contact region is significantly lower than at the absorber front surface.

[Promyelocytic leukaemia protein and defect in transforming growth factor-beta signal pathway in acute promyelocytic leukaemia]. / Protein promyelocytární leukémie a porucha signální dráhy transformacního rustového faktoru-beta u akutní promyelocytární leukémie.

Chromosome translocations are detected in 50-70% of human leukaemia. The promyelocytic leukaemia (PML) gene is involved in the t(15;17) chromosomal translocation of acute promyelocytic leukaemia (APL). PML gene encodes a protein, which was shown to be concentrated in PML-nuclear bodies. Histone acetyltransferases and deacetylases, and chromatin-modifying proteins are accumulated in complexes with PML protein in these nuclear bodies giving the evidence of their role in transcription regulation. Physical interactions of PML protein with transcription factors, co-activators and co-repressors of transcription correspond with the role of PML in transcription regulation. PML plays an important role in apoptosis, proliferation and senescence of cells. PML gene is a tumour-suppressor gene and a product of its expression acts as a potent cell growth suppressor. All these activities of PML protein are ascribed to its nuclear functions. Cytoplasmic form of PML (cPML) is also very important and it is critical for transforming growth factor-beta (TGF-beta) signalling. Cytoplasmic PML interacts with two TGF-beta receptors (TbetaBRI and TbetaRII) and acts as a bridging factor between protein called Smad anchor of receptor activation (SARA) and Smad proteins and it plays a role in the transport of whole complex into the early endosomes in TGF-beta signalling. The loss of functional cPML induces not only APL but it might influence behaviour of cancer cells and their resistance to TGF-beta.

Bonding and structure of glycine on ordered Al2O3 film surfaces.

The interaction between glycine (NH2CH2COOH) layers and an ultrathin Al2O3 film grown epitaxially onto NiAl(110) was studied by temperature-programmed desorption, X-ray photoelectron spectroscopy, ultraviolet photoelectron spectroscopy, work function measurements, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. At monolayer coverages at 110 K, there are two coexisting molecular forms: the anionic (NH2CH2COO-) and the zwitterionic form (NH3+CH2COO-) of glycine. As deduced from the photoemission data, the buildup of multilayers at 110 K leads to a condensed phase predominantly in the zwitterionic state. In contrast to the monolayer at 110 K, the monolayer formed at 300 K consists primarily of glycine molecules in the anionic state. The latter species is adsorbed with the oxygen atoms of the carboxylic group pointing toward the substrate. The polarization-dependent C K- and O K-edge NEXAFS spectra indicate that the glycinate species in the monolayer at 300 K is oriented nearly perpendicular to the surface, with the amino group pointing away from the surface.