Generating GBX2 antibodies: A useful tool in determining developmental mechanisms regulated by GBX2 [abstract]

Abstract only availableThe GBX class of homeobox genes is comprised of Gbx1 and Gbx2. Both loss-of-function and gain-of-function studies in mice have shown that Gbx2 is vital for normal anterior hindbrain development of mammalian organisms. To gain more insight into the developmental mechanisms regulated by GBX2, we are generating GBX2 antibodies. To accomplish this, we have subcloned Gbx2 into the pRSET A protein expression vector and transformed the construct into BL21(DE3)pLysS cells. Protein expression was induced by IPTG. The expressed protein was analyzed by SDS-PAGE as well as Western analysis. The purified protein will be used to elicit an immune response in chickens to generate the antibodies against GBX2. Preliminary results from the SDS-PAGE and Western analysis have suggested that the GBX2 protein is being expressed. However, further testing is necessary for confirmation. Currently, we are using Western analysis to specifically target the 6x His tagged GBX2 fusion protein in order to identify the protein for further analysis by mass spectroscopy. Generation of GBX2 antibodies will provide an important tool to enhance our knowledge of how GBX2 functions in development. Having these antibodies will allow for cellular localization. In addition, the antibodies will be used in chromatin immunoprecipitation assays, which will allow for the production of a library that contains genes directly regulated by GBX2. The identification of target genes will provide a way to enable the collection of valuable data that will be useful for more long-term research goals involving the specific signaling and genetic pathways in which this transcription factor is involved

Measurements of Proton, Helium and Muon Spectra at Small Atmospheric Depths with the BESS Spectrometer

The cosmic-ray proton, helium, and muon spectra at small atmospheric depths of 4.5 -- 28 g/cm^2 were precisely measured during the slow descending period of the BESS-2001 balloon flight. The variation of atmospheric secondary particle fluxes as a function of atmospheric depth provides fundamental information to study hadronic interactions of the primary cosmic rays with the atmosphere.Comment: 21 pages, 11 figures, 4 table

Genome-wide association study identifies multiple susceptibility loci for craniofacial microsomia

Craniofacial microsomia (CFM) is a rare congenital anomaly that involves immature derivatives from the first and second pharyngeal arches. The genetic pathogenesis of CFM is still unclear. Here we interrogate 0.9 million genetic variants in 939 CFM cases and 2,012 controls from China. After genotyping of an additional 443 cases and 1,669 controls, we identify 8 significantly associated loci with the most significant SNP rs13089920 (logistic regression P 1�4 2.15 � 10 � 120) and 5 suggestive loci. The above 13 associated loci, harboured by candidates of ROBO1, GATA3, GBX2, FGF3, NRP2, EDNRB, SHROOM3, SEMA7A, PLCD3, KLF12 and EPAS1, are found to be enriched for genes involved in neural crest cell (NCC) development and vasculogenesis. We then perform whole-genome sequencing on 21 samples from the case cohort, and identify several novel loss-of-function mutations within the associated loci. Our results provide new insights into genetic background of craniofacial microsomia

Genome-wide exploration of direct Gastrulation Brain Homeobox 2 target genes and their contributions to mouse development

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The cells that comprise the vertebrate nervous system require molecular cues to determine their cellular position, identity and their ability to make appropriate connections with their target tissues. These requirements largely rely on the precise spatial-temporal regulation of gene expression and can be controlled by a variety of intracellular mechanisms. However, a critical regulatory step in controlling gene expression is the initial transcription of the gene from an organism's genome. Transcription factors are proteins that primarily function by recognizing direct target gene sequences and modulating their expression. As such, the investigation of genes directly targeted and regulated by transcription factors provides a fundamental link in addressing their contributions to vertebrate nervous system development. There are many known classes of transcription factors encoded by the genomes of vertebrate species. One class critical for vertebrate development is the homeobox transcription factors, which encode a highly conserved DNAbinding homeodomain. The Gastrulation brain homeobox (Gbx) genes consists of two known family members, Gbx1 and Gbx2. To date, previous Gbx2 misexpression studies in the mouse have largely ascribed requirements for Gbx2 in development of the hindbrain, spinal cord, ear and heart. However, prior to the work presented in this dissertation, the direct targets and molecular pathways mediated by GBX2 were largely unknown. In order to dramatically expand our knowledge of Gbx2 function, we conducted the first genome-wide investigation of direct GBX2 target genes. Our study identified over 1,000 target genes. Applying stringent selection criteria to the identified targets, we reduced the number to 286 target genes for subsequent analyses. Interestingly, 51% of GBX2 targets identified are expressed in the nervous system. Our studies have revealed that GBX2 binds to the promoter or intronic sequences of several targets including EEF1A1, NRP1, PLXNA4, ROBO1, PCDH15, USH2A and NOTCH2. The target genes PCDH15, USH2A and NOTCH2 are involved with inner ear development. PCDH15 and USH2A are also associated with the congenital disease Usher syndrome, a condition that is characterized by the progressive loss of hearing and sight. We further demonstrated that GBX2 interacts with the EEF1A1 core promoter and functions as a transcriptional activator within this region. A primary function of EEF1A1 is the delivery of aminoacyl-tRNA to the ribosome during protein synthesis. The impact of GBX2 regulating EEF1A1 may suggest a novel GBX2-mediated mechanism for regulating protein expression during vertebrate development. The development of the anterior hindbrain is dependent on the ability of divergent motor neuron and multipotent cranial neural crest (NC) cells to migrate to their target locations. During development, the hindbrain is transiently segmented into eight compartments known as rhombomeres. How Gbx2 impacts motor neuron, cranial NC cell development and gene expression within the hindbrain is not well understood. Loss-of-function studies in zebrafish and mouse have demonstrated a requirement of Gbx2 in motor neuron development in the anterior hindbrain and spinal cord. Here we show that a loss of Gbx2 and anterior hindbrain tissue results in a disruption of r2 motor neuron development and suggest a novel requirement of Gbx2 in the correct temporal repression of the transcription factor Krox20 in r3. Cranial NC cells originating in the hindbrain are highly motile and require coordinated inputs from divergent signaling pathways to ensure their appropriate positions in the developing embryo. Many of the defects in Gbx2 mutants are observed in NC-derived structures. Furthermore, the cardiac and craniofacial phenotypes observed in Gbx2 mutants are reminiscent of defects reported in individuals with the congenital disease DiGeorge syndrome. Interestingly, GBX2 target genes ROBO1 and NRP1 are involved with NC cell migration. In mice, loss of Robo1 or Nrp1 results in the disrupted migration of NC cell subpopulations within the developing heart and anterior hindbrain. Previous studies in Gbx2-/- mice have demonstrated that a loss of Robo1 disrupts cardiac NC cell migration and contributes to the observed arterial defects. Here we provide evidence that GBX2 functions directly upstream of ROBO1 and NRP1. Data presented in this dissertation further show a loss of Robo1 and a reduction of migrating cranial NC cells from r4 in Gbx2-/- mice. Additionally, studies in Gbx2 mutant mice revealed a loss of Nrp1 and an increase in apoptosis in a subpopulation of cranial NC cells from r2. Loss of Robo1 and Nrp1 are now thought to contribute to the NC cell migratory defects and subsequently the disrupted NC-derived structures observed in Gbx2 mutant mice. These findings have led to new insights into Gbx2 function during vertebrate development. Studies discussed in this dissertation have resulted in a dramatic increase in the number of known direct GBX2 target genes and molecular pathways potentially regulated by GBX2. The subset of GBX2 targets investigated thus far suggests that they may contribute to the development of tissues and structures impacted by the loss of Gbx2 in the mouse heart, ear and hindbrain. The involvement of GBX2 and direct target genes with multiple congenital diseases further illustrates the biological and clinical importance of the findings presented in the following body of work. Future studies aimed at elucidating the biological impact of GBX2 through direct target genes will reveal the precise molecular pathways impacted by GBX2 during vertebrate development.Includes bibliographical references (pages 166-186)

Paukenerguss beim allergischen Kind – primär konservativ behandeln oder ist eine operative Sanierung erforderlich?

Der chronische Paukenerguss ist eine der häufigsten Erkrankungen im Kindesalter und hat direkte Konsequenzen für den Spracherwerb und die weitere kognitive Entwicklung eines Kindes. Für die Entstehung des Paukenergusses ist zumeist eine anhaltende Tubenventilationsstörung verantwortlich. Lokale inflammatorische Reize, die tubare Insuffizienz und der daraus resultierende tympanale Unterdruck bedingen einen zunächst serösen Erguss, der bei länger bestehenden Prozessen zunehmend mukös wird. Obwohl die derzeit in Überarbeitung befindliche deutsche Leitlinie klare Empfehlungen für konservative und chirurgische Therapie ausspricht, ist die Datenbasis für diese Empfehlungen nicht optimal, da die Patienten nicht nach klinisch relevanter Allergie stratifiziert wurden. Deswegen sind konklusive Empfehlungen für diese Patienten schwierig

Gbx2 Is Required for the Migration and Survival of a Subpopulation of Trigeminal Cranial Neural Crest Cells

The development of key structures within the mature vertebrate hindbrain requires the migration of neural crest (NC) cells and motor neurons to their appropriate target sites. Functional analyses in multiple species have revealed a requirement for the transcription factor gastrulation-brain-homeobox 2 (Gbx2) in NC cell migration and positioning of motor neurons in the developing hindbrain. In addition, loss of Gbx2 function studies in mutant mouse embryos, Gbx2neo, demonstrate a requirement for Gbx2 for the development of NC-derived sensory neurons and axons constituting the mandibular branch of the trigeminal nerve (CNV). Our recent GBX2 target gene identification study identified multiple genes required for the migration and survival of NC cells (e.g., Robo1, Slit3, Nrp1). In this report, we performed loss-of-function analyses using Gbx2neo mutant embryos, to improve our understanding of the molecular and genetic mechanisms regulated by Gbx2 during anterior hindbrain and CNV development. Analysis of Tbx20 expression in the hindbrain of Gbx2neo homozygotes revealed a severely truncated rhombomere (r)2. Our data also provide evidence demonstrating a requirement for Gbx2 in the temporal regulation of Krox20 expression in r3. Lastly, we show that Gbx2 is required for the expression of Nrp1 in a subpopulation of trigeminal NC cells, and correct migration and survival of cranial NC cells that populate the trigeminal ganglion. Taken together, these findings provide additional insight into molecular and genetic mechanisms regulated by Gbx2 that underlie NC migration, trigeminal ganglion assembly, and, more broadly, anterior hindbrain development

Evaluation of oxygen uptake and delivery in critically ill patients: A statistical reappraisal

Objective: The evaluation of oxygen consumption (VO2) and oxygen delivery (DO2) has gained increasing importance in the monitoring of critically ill patients. They can be obtained from either direct measurements or by indirect calculations based on the Fick principle. However the choice between these two approaches remains controversial. The aim of the study was to investigate whether these 2 methods provide similar results, and if not, to define the best one in terms of reproducibility. Design: Oxygen delivery and oxygen consumption were prospectively analyzed in 171 consecutive critically ill patients. Metabolic data were obtained simultaneously. Setting: The study was completed in the intensive care unit as part of the management of the patients studied. Patients: A first ''group'' of 279 evaluations was carried out in 73 consecutive critically ill patients. The results were subsequently validated by 423 observations performed in the 98 following patients. Interventions: Before and during each evaluation, the patients were kept in stable hemodynamic and metabolic conditions. All were mechanically ventilated. Measurements and results: VO2 was evaluated by calculation (Fick principle) and direct measurement using indirect calorimetry Cardiac output was both measured by the thermodilution technique and calculated (Fick principle) and the data were used for the evaluation of the directly measured and indirectly calculated DO2. For both VO2 DO2 the agreement between direct and indirect evaluations was not satisfactory. Differences as great as 55 ml/min.m(2) and 267 ml/min.m(2) between simultaneously measured and calculated VO2 and DO2 respectively may be expected. Finally, the indirect calculated methods were less reproducible than the measured ones. These observations resulted mainly from the cumulative effects of the random errors in the metabolic data entering into the calculation of VO2 and DO2. Conclusions: Our data suggested that the indirect calculation (Fick equation) and the direct measurement (indirect calorimetry, thermodilution) of both VO2 and DO2 did not provide similar results. Direct measurements are more reproducible methods and must be preferred

Significance of pathologic oxygen supply dependency in critically ill patients: Comparison between measured and calculated methods

Objective: oxygen supply dependency at normal or high oxygen delivery rate has been increasingly proposed as a hallmark and a risk factor in critical illnesses. We hypothesized that as fas as an adequate oxygen delivery is provided, oxygen consumption, when determined by indirect calorimetry, is not dependent on oxygen delivery in critically ill patients whereas calculated oxygen consumption is associated with artefactual correlation of oxygen consumption and delivery. Design: oxygen delivery, oxygen consumption and their relationship were analyzed prospectively. Metabolic data gained from both measured and calculated methods were obtained simultaneously before and after volume loading. Setting: the study was completed in the intensive care unit as part of the management protocol of the patients. Patients: 32 consecutive patients entered the study and were divided into 3 groups according to a clinical condition known to favour oxygen supply dependency: sepsis syndrome, adult respiratory distress syndrome and acute primary liver failure. Intervention: the rise in oxygen delivery was obtained by colloid infusion (oxygen flux test) performed in hemodynamically and metabolically stable patients. All were mechanically ventilated. No change in therapy was allowed during the test. Measurements and results: oxygen consumption was simultaneously evaluated by calculation (Fick Principle) and direct measurement using indirect calorimetry. Oxygen delivery was derived from the cardiac output (thermodilution) and arterial content of oxygen. Oxygen supply dependency was considered while observing an increase in oxygen delivery greater than 45 ml/min.m(2). Irrespective of patient's clinical diagnosis and outcome, measured oxygen uptake remained unaltered by volume infusion whereas both oxygen delivery and calculated oxygen consumption increased significantly. Arterial lactate level>2mmol/l and measured oxygen extraction ratio>25% failed to identify oxygen supply dependency when measured data were considered. Conclusion: analysis of oxygen uptake, when measured by indirect calorimetry, failed to substantiate oxygen supply dependency in the vast majority of the critically ill patients irrespective of diagnosis and outcome. Mathematical coupling of shared variables accounted for the correlation between oxygen delivery and calculated oxygen consumption