Entwicklung innovativer Trägersysteme zur gezielten dermalen Applikation eines GATA-3-spezifischen DNAzyms als Therapeutikum der Atopischen Dermatitis

Da die akute Phase der Atopischen Dermatitis mit einer Überexpression von Th2-Zellen einhergeht und der Transkriptionsfaktor GATA-3 hierfür einen entscheidenden Trigger darstellt, ist es naheliegend, durch ein selektives Ausschalten der mRNA die Expression dieses Transkriptionsfaktors zu unterbinden und somit die Erkrankung spezifisch und kausal zu therapieren. Eine Möglichkeit zur spezifischen Katalyse von GATA-3 stellt das Antisense Oligonukleotid DNAzym hgd 40 dar. Hauterkrankungen, wie die Atopische Dermatitis werden konventionell dermal therapiert. Allerdings ergeben sich für die bis dato noch nicht in die dermale Therapie eingeführten Antisense Oligonukleotide aufgrund von Molekülgröße und Sensitivität zahlreiche Herausforderungen. Besonders gegenüber in und auf der Haut ubiquitär vorkommenden DNasen müssen diese Wirkstoffe vor Degradation geschützt werden. Gleichzeitig ist eine ausreichende Resorption in das Zielorgan Haut sicherzustellen. Folgerichtig wird in der vorliegenden Arbeit die Formulierungsfindung für den als Modellsubstanz ausgewählten Wirkstoff DNAzym hgd 40 behandelt. Hierbei war des besonders zu beachten, dass die natürliche Hautbarriere bei der Atopischen Dermatitis zwar geschwächt, gleichzeitig aber auch außerhalb eines Krankheitsschubs bei intakter Hautbarriere die Wirkstoffpenetration für einen präventiven Effekt gewährleistet sein sollte. Multiple Emulsionen wurden als Kandidat mit der größten Schnittmenge an Eigenschaften, wie Wirkstoffschutz und Wirkstoffpenetration bei gleichzeitig pflegenden Eigenschaften, favorisiert. Multiple Emulsionen sind Wasser-in-Öl-in-Wasser Emulsionen, in deren innerer Wasserphase Wirkstoffe eingeschlossen werden können. Zum Vergleich wurden weitere Emulsionstypen, wie Mikroemulsion, Submikronemulsion und konventionelle Wasser-in-Öl-Emulsionen in die Versuchsreihe aufgenommen. Es konnte gezeigt werden, dass alle untersuchten Emulsionstypen hinsichtlich ihrer physikochemischen Parameter, wie pH-Wert, Tropfengröße und Viskosität über den untersuchten Zeitraum stabil sind. Ein besonderes Augenmerk richtete sich auf die Schutzfunktion der Galeniken gegenüber der Degradation des Wirkstoffs DNAzym hgd 40. Die hierzu notwendigen extraktions- und analytischen Verfahren konnten im Rahmen dieser Arbeit etabliert werden. Bei Inkubationsversuchen mit hautidentischen DNasen wurde gezeigt, dass multiple Emulsionen innerhalb des untersuchten Zeitraums über 50% des in der inneren Phase eingearbeiteten Wirkstoffs effektiv vor Abbau schützen. Die beschriebenen Trägersysteme Mikroemulsion und Submikronemulsion zeigten keine Schutzfunktion und einen nahezu vollständigen Abbau des Wirkstoffs. Zur Untersuchung der Kinetik wurden Freisetzungsversuche an Cellulosemembranen durchgeführt. Die positiven Ergebnisse dieser Arbeit lassen eine kausale, dermale Therapie der Atopischen Dermatitis mit Antisense Oligonukleotiden, geschützt in multiplen Emulsionen, in greifbare Nähe rücken

The ancient harbour city of Ainos and its environs – Palaeoenvironmental reconstructions based on sedimentological and microfaunal evidence

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Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. We demonstrate that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, we demonstrate that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. Our findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes

Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects.

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering

Two-pion Bose-Einstein correlations in central Pb-Pb collisions at sNN\sqrt{s_{\rm NN}} = 2.76 TeV

The first measurement of two-pion Bose-Einstein correlations in central Pb-Pb collisions at $\sqrt{s_{\rm NN}} = 2.76$ TeV at the Large Hadron Collider is presented. We observe a growing trend with energy now not only for the longitudinal and the outward but also for the sideward pion source radius. The pion homogeneity volume and the decoupling time are significantly larger than those measured at RHIC.Comment: 17 pages, 5 captioned figures, 1 table, authors from page 12, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/388

Suppression of charged particle production at large transverse momentum in central Pb-Pb collisions at sNN=2.76\sqrt{s_{\rm NN}} = 2.76 TeV

Inclusive transverse momentum spectra of primary charged particles in Pb-Pb collisions at $\sqrt{s_{_{\rm NN}}}$ = 2.76 TeV have been measured by the ALICE Collaboration at the LHC. The data are presented for central and peripheral collisions, corresponding to 0-5% and 70-80% of the hadronic Pb-Pb cross section. The measured charged particle spectra in $|\eta|<0.8$ and $0.3 < p_T < 20$ GeV/$c$ are compared to the expectation in pp collisions at the same $\sqrt{s_{\rm NN}}$, scaled by the number of underlying nucleon-nucleon collisions. The comparison is expressed in terms of the nuclear modification factor $R_{\rm AA}$. The result indicates only weak medium effects ($R_{\rm AA} \approx$ 0.7) in peripheral collisions. In central collisions, $R_{\rm AA}$ reaches a minimum of about 0.14 at $p_{\rm T}=6$-7GeV/$c$ and increases significantly at larger $p_{\rm T}$. The measured suppression of high-$p_{\rm T}$ particles is stronger than that observed at lower collision energies, indicating that a very dense medium is formed in central Pb-Pb collisions at the LHC.Comment: 15 pages, 5 captioned figures, 3 tables, authors from page 10, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/98

Expanding the clinical phenotype of IARS2-related mitochondrial disease.

BACKGROUND: IARS2 encodes a mitochondrial isoleucyl-tRNA synthetase, a highly conserved nuclear-encoded enzyme required for the charging of tRNAs with their cognate amino acid for translation. Recently, pathogenic IARS2 variants have been identified in a number of patients presenting broad clinical phenotypes with autosomal recessive inheritance. These phenotypes range from Leigh and West syndrome to a new syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia, as well as cataract with no additional anomalies. METHODS: Genomic DNA from Iranian probands from two families with consanguineous parental background and overlapping CAGSSS features were subjected to exome sequencing and bioinformatics analysis. RESULTS: Exome sequencing and data analysis revealed a novel homozygous missense variant (c.2625C > T, p.Pro909Ser, NM_018060.3) within a 14.3 Mb run of homozygosity in proband 1 and a novel homozygous missense variant (c.2282A > G, p.His761Arg) residing in an ~ 8 Mb region of homozygosity in a proband of the second family. Patient-derived fibroblasts from proband 1 showed normal respiratory chain enzyme activity, as well as unchanged oxidative phosphorylation protein subunits and IARS2 levels. Homology modelling of the known and novel amino acid residue substitutions in IARS2 provided insight into the possible consequence of these variants on function and structure of the protein. CONCLUSIONS: This study further expands the phenotypic spectrum of IARS2 pathogenic variants to include two patients (patients 2 and 3) with cataract and skeletal dysplasia and no other features of CAGSSS to the possible presentation of the defects in IARS2. Additionally, this study suggests that adult patients with CAGSSS may manifest central adrenal insufficiency and type II esophageal achalasia and proposes that a variable sensorineural hearing loss onset, proportionate short stature, polyneuropathy, and mild dysmorphic features are possible, as seen in patient 1. Our findings support that even though biallelic IARS2 pathogenic variants can result in a distinctive, clinically recognisable phenotype in humans, it can also show a wide range of clinical presentation from severe pediatric neurological disorders of Leigh and West syndrome to both non-syndromic cataract and cataract accompanied by skeletal dysplasia