Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development.

BACKGROUND: The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. RESULTS: In this study we conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. We generated a new hbnKOGal4 enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. We characterized several enhancers that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, we generated deletions of four of these enhancer regions through gene targeting and analysed their effects on the expression and function of hbn. CONCLUSION: The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.

Functional analysis of enhancer elements regulating the expression of the Drosophila homeodomain transcription factor DRx by gene targeting.

BACKGROUND: The Drosophila brain is an ideal model system to study stem cells, here called neuroblasts, and the generation of neural lineages. Many transcriptional activators are involved in formation of the brain during the development of Drosophila melanogaster. The transcription factor Drosophila Retinal homeobox (DRx), a member of the 57B homeobox gene cluster, is also one of these factors for brain development. RESULTS: In this study a detailed expression analysis of DRx in different developmental stages was conducted. We show that DRx is expressed in the embryonic brain in the protocerebrum, in the larval brain in the DM and DL lineages, the medulla and the lobula complex and in the central complex of the adult brain. We generated a DRx enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the endogenous expression of DRx. With the help of eight existing enhancer-Gal4 strains and one made by our group, we mapped various enhancers necessary for the expression of DRx during all stages of brain development from the embryo to the adult. We made an analysis of some larger enhancer regions by gene targeting. Deletion of three of these enhancers showing the most prominent expression patterns in the brain resulted in specific temporal and spatial loss of DRx expression in defined brain structures. CONCLUSION: Our data show that DRx is expressed in specific neuroblasts and defined neural lineages and suggest that DRx is another important factor for Drosophila brain development.

The homeodomain transcription factor Orthopedia is involved in development of the Drosophila hindgut.

BACKGROUND: The Drosophila hindgut is commonly used model for studying various aspects of organogenesis like primordium establishment, further specification, patterning, and morphogenesis. During embryonic development of Drosophila, many transcriptional activators are involved in the formation of the hindgut. The transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, is expressed in the hindgut and nervous system of developing Drosophila embryos, but due to the lack of mutants no functional analysis has been conducted yet. RESULTS: We show that two different otp transcripts, a hindgut-specific and a nervous system-specific form, are present in the Drosophila embryo. Using an Otp antibody, a detailed expression analysis during hindgut development was carried out. Otp was not only expressed in the embryonic hindgut, but also in the larval and adult hindgut. To analyse the function of otp, we generated the mutant otp allele otpGT by ends-out gene targeting. In addition, we isolated two EMS-induced otp alleles in a genetic screen for mutants of the 57B region. All three otp alleles showed embryonic lethality with a severe hindgut phenotype. Anal pads were reduced and the large intestine was completely missing. This phenotype is due to apoptosis in the hindgut primordium and the developing hindgut. CONCLUSION: Our data suggest that Otp is another important factor for hindgut development of Drosophila. As a downstream factor of byn Otp is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells.

Roughening and long-range nanopatterning of Au(111) through potential cycling in aqueous acidic media.

Electrochemical treatment of Au(111) in aqueous H2SO4 solution by repetitive application of oxide formation-reduction cycles (OFRC) generates nanopatterned surfaces with long-range order. The pattern development depends on the lower and upper potential limits (EL, EU), the number (n) of OFRCs, and the potential scan rate (s). Surface patterning of Au(111) initially (n = 1-2) generates small islands and holes that are one atomic step in height. As n increases to 5, the number of islands decreases and the holes become larger; after n = 10 OFRCs, the islands become inexistent and large, randomly distributed holes are observed. Increase of OFRCs to n = 20 generates surface structures that reside within three atomic layers and resemble phase separation through a spinodal decomposition mechanism. As the number of OFRCs rises to n = 50, a network of interconnected islands and holes emerges; the islands and holes are two-three atomic steps in height, and are located within topmost five monolayers. Further increase of the number of OFRCs to n = 100 creates a network of interconnected trigonal pyramids that are pointed in the same direction. The size of the pyramids depends on the electrolyte composition and the number of OFRCs. In the case of n = 100, the pyramids are 12-25 nm in base length and 0.4-1.6 nm in height in 0.1 M aqueous H2SO4, and 20-50 nm in base length and 0.8-1.6 nm in height in 0.1 M aqueous HNO3. The number of OFRCs and scan rate play an important role in patterning of Au(111), and complete nanopattern development requires a large number of OFRCs and low scan rates.

The interfaces of Au(111) and Au(100) in a hexaalkyl-substituted guanidinium ionic liquid: an electrochemical and in situ STM study.

Five hexaalkylguanidinium-based ionic liquids have been synthesised, and based on their cyclic voltammograms the most suited one, N,N-dibutyl-N',N'-diethyl-N'',N''-dimethylguanidinium bis(trifluoromethylsulfonyl)imide, has been chosen for electrochemical studies. The surface interaction of this room-temperature ionic liquid with single crystalline gold surfaces (Au(100) and Au(111)) has been investigated using cyclic voltammetry, impedance spectroscopy and in situ scanning tunnelling microscopy (STM). The interfacial capacitance was found to be very low; STM measurements revealed the hex-reconstruction and herringbone reconstruction for Au(100) and for Au(111), respectively, at negative potentials; that is, at these potentials no hints for ad-structures of the cation could be found.

The Drosophila homeodomain transcription factor Homeobrain is involved in the formation of the embryonic protocerebrum and the supraesophageal brain commissure.

During the embryonic development of Drosophila melanogaster many transcriptional activators are involved in the formation of the embryonic brain. In our study we show that the transcription factor Homeobrain (Hbn), a member of the 57B homeobox gene cluster, is an additional factor involved in the formation of the embryonic Drosophila brain. Using a Hbn antibody and specific cell type markers a detailed expression analysis during embryonic brain development was conducted. We show that Hbn is expressed in several regions in the protocerebrum, including fibre tract founder cells closely associated with the supraesophageal brain commissure and also in the mushroom bodies. During the formation of the supraesophageal commissure, Hbn and FasII-positive founder cells build an interhemispheric bridge priming the commissure and thereby linking both brain hemispheres. The Hbn expression is restricted to neural but not glial cells in the embryonic brain. In a mutagenesis screen we generated two mutant hbn alleles that both show embryonic lethality. The phenotype of the hbn mutant alleles is characterized by a reduction of the protocerebrum, a loss of the supraesophageal commissure and mushroom body progenitors and also by a dislocation of the optic lobes. Extensive apoptosis correlates with the impaired formation of the embryonic protocerebrum and the supraesophageal commissure. Our results show that Hbn is another important factor for embryonic brain development in Drosophila melanogaster.

Metallization of ultra-thin, non-thiol SAMs with flat-lying molecular units: Pd on 1, 4-dicyanobenzene.

Self-assembled monolayers of 1,4-dicyanobenzene on Au(111) electrodes are studied by cyclic voltammetry, in-situ STM and ex-situ XPS. High-resolution STM images reveal a long-range order of propeller-like assemblies each of which consists of three molecules, all lying flat on the gold substrate with the cyano groups oriented parallel to the metal surface. It is demonstrated that both functional groups can act as complexation sites for metal ions from solution. Surprisingly, such arrangements still allow the metal to be deposited on top of the molecules by electrochemical reduction despite the close vicinity to the Au surface. The latter is demonstrated by angle-resolved XPS which unequivocally shows that the metal indeed resides on top of the organic layer rather than underneath, despite the flat arrangement of the molecules.

Metallization of organic surfaces: Pd on thiazole.

Results from electrochemical studies, in situ STM, and ex situ angle-resolved XPS on self-assembled monolayers (SAMs) of thiazole on Au(111) are reported for the first time. Although STM seems to indicate a low density of molecules organized in small ordered domains, a quantitative chemical analysis of the sample surface by XPS clearly points toward the formation of a densely packed molecular layer. The stability of the thiazole SAM against reductive desorption is found to be very comparable with that for thiol-SAMs on gold. This results from the formation of Au-S bonds between the molecules and their support as evidenced by XPS, thereby rebuting speculations that the ring nitrogen is responsible for the attachment of such molecules to gold surfaces. Consequently, the N-atoms terminating the molecular layer are available as active sites for the complexation with Pd ions thereby allowing the deposition of Pd islands with monatomic height on top of the thiazole SAM. The importance of such studies for metal-molecule interconnections is briefly addressed.

Enzyme immobilisation on self-organised nanopatterned electrode surfaces.

A new method is described for immobilisation of enzymes on polymer-coated Pt islands. These islands are deposited on top of a SAM-covered Au(111) electrode by a combination of electroless and electrochemical deposition, which allows for a variation of island size and distance between the islands. Here we describe the immobilisation of pyranose-2-oxidase (P2Ox) and the catalytic response to D-glucose on such a nanopatterned surface, which provides optimum access to the active centres of the enzyme.

Retinal homeobox promotes cell growth, proliferation and survival of mushroom body neuroblasts in the Drosophila brain.

The Drosophila mushroom bodies, centers of olfactory learning and memory in the fly 'forebrain', develop from a set of neural stem cells (neuroblasts) that generate a large number of Kenyon cells (KCs) during sustained cell divisions from embryonic to late pupal stage. We show that retinal homeobox (rx), encoding for an evolutionarily conserved transcription factor, is required for proper development of the mushroom bodies. Throughout development rx is expressed in mushroom body neuroblasts (MBNBs), their ganglion mother cells (MB-GMCs) and young KCs. In the absence of rx function, MBNBs form correctly but exhibit a reduction in cell size and mitotic activity, whereas overexpression of rx increases growth of MBNBs. These data suggest that Rx is involved in the control of MBNB growth and proliferation. Rx also promotes cell cycling of MB-GMCs. Moreover, we show that Rx is important for the survival of MBNBs and Kenyon cells which undergo premature cell death in the absence of rx function. Simultaneous blocking of cell death restores the normal set of MBNBs and part of the KCs, demonstrating that both, impaired proliferation and premature cell death (of MBNBs and KCs) account for the observed defects in mushroom body development. We then show that Rx controls proliferation within the MBNB clones independently of Tailless (Tll) and Prospero (Pros), and does not regulate the expression of other key regulators of MB development, Eyeless (Ey) and Dachshund (Dac). Our data support that the role of Rx in forebrain development is conserved between vertebrates and fly.

A multidisciplinary quality management system for the early treatment of severely injured patients: implementation and results in two trauma centers.

OBJECTIVE: The impact of a multidisciplinary quality management system (MQMS) on the early treatment of severely injured patients was tested. DESIGN AND SETTING: Prospective clinical study in two level 1 trauma centers. METHODS AND MATERIALS: MQMS comprised a protocol for documentation, 20 assessment criteria, and the judgement of data by a quality circle. After implementation in Munich (1st period, n=90; 2nd period, n=77) the validation took place in Essen (1st period, n=175; 2nd period, n=150). RESULTS: Improvements in diagnostics were shown by significant time savings in radiological diagnostics and before computed tomography in severe traumatic brain injury. In patients with hemorrhagic shock there was a reduction in time before transfusion (49 to 14 min in Munich; 31 to 22 min in Essen) and before emergency operation (74 to 43 min in Munich; 69 to 45 min in Essen). The time before craniotomy was reduced from 97 to 67 min in Munich. The incidence of delayed diagnosis of life-threatening lesions was diminished from 6% to 3% in Munich (not found in Essen). The TRISS technique showed a reduction in mortality in both hospitals in the second period (Munich: 15.4% TRISS vs. 9.1% observed mortality; Essen: 17.8% vs. 11.3%). CONCLUSIONS: MQMS improved early clinical treatment in severe injury with respect to therapeutic effectiveness and outcome. The effectiveness of the MQMS was shown at two different hospitals

Human leukocyte antigen-DR expression in peripheral blood mononuclear cells from healthy donors influenced by the sera of injured patients prone to severe sepsis.

OBJECTIVE: To study the influence of sera from severely injured patients on the human leukocyte antigen (HLA)-DR expression of normal peripheral blood mononuclear cells (PBMC). DESIGN: In vitro study. SETTING: University hospital. PATIENTS AND PARTICIPANTS: Sera from 34 patients were obtained within 8 h after trauma. Seventeen of these patients developed posttraumatic sepsis (sepsis group) and 17 recovered without infectious complications. Sera from ten healthy individuals served as controls. Phytohemagglutinin (PHA)-activated PBMC from 44 healthy donors were used to study the effects of a patient's serum. MEASUREMENTS AND RESULTS: Medium containing 5% of serum from the sepsis group significantly ( p<0.05) reduced the HLA-DR expression (channels, mean +/- standard error of the mean) on monocytes (patients 883+/-22, controls 962+/-15), B (patients 922+/-14, controls 972+/-7) and T cells (patients 932+/-13, controls 968+/-5) of PHA-activated PBMC. Significantly increased accumulation of TNFalpha on (1.8+/-0.4% of PBMC) and within T cells (0.98+/-0.26% of PBMC) was observed by flow cytometry after incubation with medium containing sera of the sepsis group compared with controls (on 0.5+/-0.1%, within 0.27+/-0.05% of PBMC). A significant negative correlation between relative cell counts of intracellular TNFalpha-positive T cells with HLA-DR expression was observed for monocytes ( r= -0.61), B cells ( r= -0.57) and proliferation ( r= -0.68) as estimated by (3)H-thymidine uptake [patients 139971+/-12844 counts per minute (cpm), controls 198973+/-19347 cpm, p<0.05] in the presence of sera from the sepsis group. CONCLUSIONS: Reduced cellular immunity and, therefore, immunodeficiency after trauma appears to be caused by soluble factors influencing T cell function in particular.

Electrochemical Surface Science This manuscript is based on the Bonhoeffer-Eucken-Scheibe lectures of the Deutsche Bunsengesellschaft, given by the author at Erlangen, Berlin, and Leipzig in 1999/2000.

The last 30 years have seen remarkable changes in interfacial electrochemistry, particularly in the kind of questions that were addressed in electrochemical studies. Ever since classical surface science, traditionally performed under ultrahigh vacuum conditions, has succeeded in describing surfaces and surface reactions on a molecular level, electrochemists longed for a microscopic understanding of the solid/electrolyte interface and, at the same time, searched widely for new experimental ways to reach that goal. Herein, studies are described concerning the structure and the dynamics of bare and adsorbate-covered electrode surfaces and of metal deposition as a simple, yet important, electrochemical process. In all these cases, the scanning tunneling microscope plays a pivotal role emphasizing the surface-science approach to the problems.

Restructuring of an Ir(210) electrode surface by potential cycling.

This study addresses the electrochemical surface faceting and restructuring of Ir(210) single crystal electrodes. Cyclic voltammetry measurements and in situ scanning tunnelling microscopy are used to probe structural changes and variations in the electrochemical behaviour after potential cycling of Ir(210) in 0.1 M H2SO4. Faceted structures are obtained electrochemically as a function of time by cycling at a scanrate of 1 V·s(-1) between -0.28 and 0.70 V vs SCE, i.e., between the onset of hydrogen evolution and the surface oxidation regime. The electrochemical behaviour in sulfuric acid solution is compared with that of thermally faceted Ir(210), which shows a sharp characteristic voltammetric peak for (311) facets. Structures similar to thermally-induced faceted Ir(210) are obtained electrochemically, which typically correspond to polyoriented facets at nano-pyramids. These structures grow anisotropically in a preferred direction and reach a height of about 5 nm after 4 h of cycling. The structural changes are reflected in variations of the electrocatalytic activity towards carbon monoxide adlayer oxidation.

Prognostic factors of liver injury in polytraumatic patients. Results from 895 severe abdominal trauma cases.

BACKGROUND AND AIMS: Prognosis of multiple injured patients is mainly limited by severe haemorrhage. Although mechanisms of altered immune response have been intensively investigated, little is known about the relevance of liver trauma as an independent predictive outcome factor in these patients. METHODS: 10,469 patients from the DGU Trauma Registry (1993-2005) were retrospectively analyzed. Primary admitted patients with an injury severity score > or = 16, without isolated head injury were included. Patients were analyzed according to the injury pattern as liver injury (Abbreviated Injury Scale--AIS abdomen < 3 and AIS liver 2-5; n = 321), non-liver abdominal trauma (AIS abdomen 2-5 or AIS liver < 3; n = 574) and control group without abdominal injuries (AIS abdomen or liver < 3; n = 9,574). RESULTS: Severe liver injury was associated with excessive demands for volume resuscitation and induced a significantly increased risk for sepsis and multi-organ failure (MOF) compared to both other groups (sepsis 19.9% vs. 11.0%; MOF 32.7% vs. 16.6%). Furthermore, deleterious outcome was more frequently associated with severe liver trauma (mortality 34.9%) compared to severe abdominal trauma (12.0%). CONCLUSION: Severe liver trauma is an independent predictor for severe haemorrhage with a substantially increased risk of sepsis, MOF and trauma-related death. While conservative treatment of patients with liver trauma but no haemorrhage is effective, patients with hemodynamic instability seem to be from a subgroup where contemporary treatment modalities are not yet sufficient.