Inhibition of the Hedgehog pathway in combination with cytostatics as potential therapeutic option in Ewing Sarcoma

Ewing Sarkome (EWS) zählen zu den mesenchymalen Tumoren und treten gehäuft bei Kindern und Jugendlichen auf. Patienten mit nicht-metastasiertem EWS, die sich einer radikalen chirurgischen Intervention, Radiotherapie und/oder Chemotherapie unterziehen, zeigen eine durchschnittliche 5-Jahres Überlebensrate von über 70 %, wohingegen metastasierte und rezidivierte EWS deutlich schwerer zu behandeln sind. Einer der vielen Gründe dafür ist die zunehmende Resistenz von rezidivierten EWS gegen Chemotherapeutika. Diese beruht unter anderem darauf, dass ABC Transporter wie PGP oder MRP1, welche die Chemotherapeutika wieder aus der Tumorzelle in den Extrazellularraum befördern, von diesen exprimiert werden. Darüber hinaus wird vom EWS das für den Tumor charakteristische Fusionsprotein EWS-FLI1 exprimiert, welches auch die GLI Proteine im Hedgehog (Hh) Signalweg moduliert. Veränderungen im Hh Signalweg wurden bereits in verschiedenen Tumoren beobachtet und korrelieren mit einer erhöhten Tumorbildung und Progression. Da der Hh Signalweg direkt die Expression von ABC Transportern beeinflusst, wurde in dieser Arbeit die Inhibition der GLI Proteine als mögliche zusätzliche Therapieoption zur Unterdrückung der Chemoresistenz getestet. Dafür wurden drei EWS Zelllinien mit den GLI Inhibitoren GANT61 und ATO, sowie den bereits etablierten Chemotherapeutika Etoposid und Doxorubicin behandelt und ihre Viabilität, Koloniebildungsfähigkeit, Wachstum in 3D Kulturen und Induktion von Zelltod mittels MTS Assay, Koloniebildungs-Assay, 3D Spheroid Assay, Durchflusszytometrie und Western Blot getestet. ATO, Etoposid und Doxorubicin reduzierten die Viabilität signifikant und selektiv in allen drei EWS Zelllinien im Vergleich zu mesenchymalen Stammzellen (MSC). Dahingegen war GANT61 weniger effizient in zwei der drei EWS Zelllinien und vor allem nicht selektiv gegenüber MSC. Zelltod konnte durch ATO, Etoposid und Doxorubicin in allen EWS Zelllinien, nicht aber in MSC induziert werden. Dieser Effekt wurde mit der Kombination von ATO und Etoposid noch einmal potenziert. Die Kombination von ATO und Doxorubicin reduzierte die Viabilität ebenfalls signifikant, verglichen mit der Einzelgabe. Eine dreifache Kombination aus ATO, Etoposid und Doxorubicin zeigte jedoch keinen zusätzlichen Nutzen im Vergleich zu den Zweifach-Kombinationen. Die bisherigen Ergebnisse deuten darauf hin, dass ATO zur Erhöhung der Überlebensraten von EWS Patienten und zur Reduzierung von Resistenzen gegen Chemotherapeutika in aggressiven EWS beitragen kann. Durch die reduzierte Dosis der kombinierten Substanzen können Nebenwirkungen der Chemotherapeutika vermindert werden. Vor einem potenziellen klinischen Einsatz werden allerdings weitere Informationen über die detaillierten Wirkmechanismen in EWS benötigt.Ewing sarcoma (EWS) are mesenchymal tumours usually occurring in children and adolescents. Currently, patients with non-metastatic EWS show 5-year survival rates above 70 % when treated radically with surgery, chemotherapy and radiotherapy, whereas relapsed or metastasised EWS are more difficult to treat. One of many reasons is the increasing drug resistance in relapsed EWS including the expression of ABC transporters such as PGP or MRP1 which transport cytostatic drugs back through the cell membrane into the extracellular matrix. Furthermore, EWS express the characteristic EWS-FLI1 fusion protein which also modulates the GLI proteins in the Hedgehog (Hh) pathway. Aberrant Hh-signalling has been observed in several malignancies including EWS and correlates with cancer formation and progression. As the Hh pathway directly targets expression of ABC transporter proteins, GLI inhibition as additional therapeutic option to overcome multi drug resistance was investigated. In the present study viability, colony formation, growth of 3D cultures and induction of cell death was analysed in three EWS cell lines treated with the GLI inhibitors GANT61 and ATO as well as the established cytostatics etoposide and doxorubicin using MTS assay, clonogenic assay, 3D spheroid assay, flow cytometry and western blot. ATO, etoposide and doxorubicin reduced viability significantly and selectively in all three EWS cell lines in comparison to mesenchymal stem cells (MSC). On the other hand, GANT61 was less efficient in two of three EWS cell lines and particularly not selective compared to MSC. Cell death was induced by ATO, etoposide and doxorubicin in all three EWS cell lines, but not MSC. The combination of ATO and etoposide potentiated this effect. The combined application of ATO and doxorubicin also significantly compromised viability compared to individual treatment. However, a triple combination of ATO with both cytostatics did not compromise cells more than the double combinations. The present results indicate that ATO is effective in EWS. Therefore, ATO may contribute to increased survival rates of patients with EWS and especially reduce resistance of aggressive EWS against chemotherapeutics. Furthermore, as doses for drugs can be reduced in combined treatment, adverse effects of chemotherapy may be diminished. However, further evaluation of the detailed inhibitory mechanisms in EWS is needed before transferring this approach to the clinic

No Till and Organic Farming Improve Soil Properties but Reduce Crop Yield Compared to Conventional Farming in a Swiss Farm Network

Soils are of vital importance for sustainable food production. In order to maintain or improve soil quality, it is necessary to develop strategies for a sustainable use of soil. Alternative cropping practices such as reduced tillage and improved crop rotation are more and more adopted with the aim of decreasing the impact of agriculture on the environment. However, their on-the-ground impact in Swiss farming systems still has to be assessed. In this study, we quantified the impact of three farming systems (conventional farming, no-till, and organic farming) on plant and soil chemical, biological and physical properties. Our study included 20 fields for each farming system. All selected fields were cultivated with winter wheat the year of sampling. Soil was sampled at four layers, 0-5 cm, 5-20 cm, 20-25 cm, 25-50 cm. The main variables analysed were grain yield, soil nutrient availability, organic carbon stocks, bulk density, aggregation, porosity and soil biology. This was complemented with a comprehensive survey to collect information about cropping practices at field and farm scale, including organic matter inputs, fertilisation, tillage, phytosanitary treatments, and crop rotation.Our results show a significant influence of cropping practices on plant and soil properties. Wheat yield in no till and organic systems was reduced by 10% and 30% compared to conventional systems. Bulk density was higher in no-till than in ploughed fields in the 5-20 cm layer but similar in the subsoil. A strong stratification with depth of nutrients and soil organic carbon was observed in no-till fields. No-till and organic fields showed larger soil aggregates and higher microbial biomass in the surface layer (0-5 cm). Mycorrhizal colonisation of wheat roots was on average 50% higher in organic fields. However, no differences in carbon stock in the 0-20 cm layer was observed and the ratio organic matter / clay shows a high variability (from poor to good) and was not dependent on the farming system.Our results show that an improvement of soil properties can be achieved with alternative cropping practices such as no-till and organic farming, but also depends on the other practices adopted by the farmers, such as input of organic amendments, crop rotation diversification, residue management

Synergism between production and soil health through crop diversification, organic amendments and crop protection in wheat-based systems

One of the critical challenges in agriculture is enhancing yield without compromising its foundation, a healthy environment and, particularly, soils. Hence, there is an urgent need to identify management practices that simultaneously support soil health and production and help achieve environmentally sound production systems.To investigate how management influences production and soil health under realistic agronomic conditions, we conducted an on-farm study involving 60 wheat fields managed conventionally, under no-till or organically. We assessed 68 variables defining management, production and soil health properties. We examined how management systems and individual practices describing crop diversification, fertiliser inputs, agrochemical use and soil disturbance influenced production-quantity and quality-and soil health focusing on aspects ranging from soil organic matter over soil structure to microbial abundance and diversity.Our on-farm comparison showed marked differences between soil health and production in the current system: organic management resulted in the best overall soil health (+47%) but the most significant yield gap (-34%) compared to conventional management. No-till systems were generally intermediate, exhibiting a smaller yield gap (-17%) and only a marginally improved level of soil health (+5%) compared to conventional management. Yet, the overlap between management systems in production and soil health properties was considerably large.Our results further highlight the importance of soil health for productivity by revealing positive associations between crop yield and soil health properties, particularly under conventional management, whereas factors such as weed pressure were more dominant in organic systems.None of the three systems showed advantages in supporting production-soil health-based multifunctionality. In contrast, a cross-system analysis suggests that multifunctional agroecosystems could be achieved through a combination of crop diversification and organic amendments with effective crop protection.Synthesis and applications: Our on-farm study implies that current trade-offs in managing production and soil health could be overcome through more balanced systems incorporating conventional and alternative approaches. Such multifunctionality supporting systems could unlock synergies between vital ecosystem services and help achieve productive yet environmentally sound agriculture supported by healthy soils

Synergism between production and soil health through crop diversification, organic amendments and crop protection in wheat‐based systems

One of the critical challenges in agriculture is enhancing yield without compromising its foundation, a healthy environment and, particularly, soils. Hence, there is an urgent need to identify management practices that simultaneously support soil health and production and help achieve environmentally sound production systems. To investigate how management influences production and soil health under realistic agronomic conditions, we conducted an on‐farm study involving 60 wheat fields managed conventionally, under no‐till or organically. We assessed 68 variables defining management, production and soil health properties. We examined how management systems and individual practices describing crop diversification, fertiliser inputs, agrochemical use and soil disturbance influenced production—quantity and quality—and soil health focusing on aspects ranging from soil organic matter over soil structure to microbial abundance and diversity. Our on‐farm comparison showed marked differences between soil health and production in the current system: organic management resulted in the best overall soil health (+47%) but the most significant yield gap (−34%) compared to conventional management. No‐till systems were generally intermediate, exhibiting a smaller yield gap (−17%) and only a marginally improved level of soil health (+5%) compared to conventional management. Yet, the overlap between management systems in production and soil health properties was considerably large. Our results further highlight the importance of soil health for productivity by revealing positive associations between crop yield and soil health properties, particularly under conventional management, whereas factors such as weed pressure were more dominant in organic systems. None of the three systems showed advantages in supporting production‐soil health‐based multifunctionality. In contrast, a cross‐system analysis suggests that multifunctional agroecosystems could be achieved through a combination of crop diversification and organic amendments with effective crop protection. Synthesis and applications: Our on‐farm study implies that current trade‐offs in managing production and soil health could be overcome through more balanced systems incorporating conventional and alternative approaches. Such multifunctionality supporting systems could unlock synergies between vital ecosystem services and help achieve productive yet environmentally sound agriculture supported by healthy soils

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design

Analysis of Prototype Foamy Virus particle-host cell interaction with autofluorescent retroviral particles

<p>Abstract</p> <p>Background</p> <p>The foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive.</p> <p>Results</p> <p>In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction.</p> <p>Conclusions</p> <p>We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV - host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines resistant to Prototype Foamy Virus Env-mediated gene transfer. Interestingly, both cell lines still displayed FV Env-dependent attachment of fluorescent retroviral particles, implying a post-binding block potentially due to lack of putative FV entry cofactors. These cell lines might ultimately lead to the identification of the currently unknown ubiquitous cellular entry receptor(s) of FVs.</p

Light-driven electron injection from a biotinylated triarylamine donor to [Ru(diimine)3]2+-labeled streptavidin

Electron transfer from a biotinylated electron donor to photochemically generated Ru(III) complexes covalently anchored to streptavidin is demonstrated by means of time-resolved laser spectroscopy. Through site-selective mutagenesis, a single cysteine residue was engineered at four different positions on streptavidin, and a Ru(II) tris-diimine complex was then bioconjugated to the exposed cysteines. A biotinylated triarylamine electron donor was added to the Ru(II)-modified streptavidins to afford dyads localized within a streptavidin host. The resulting systems were subjected to electron transfer studies. In some of the explored mutants, the phototriggered electron transfer between triarylamine and Ru(III) is complete within 10 ns, thus highlighting the potential of such artificial metalloenzymes to perform photoredox catalysis

NECESSIDADE DE TRATAMENTO ODONTOLÓGICO EM PACIENTES COM TRANSTORNO DO ESPECTRO AUTISTA

O Transtorno do Espectro Autista (TEA) exige demandas variadas na área da saúde, especialmente na odontologia, os indivíduos com TEA, normalmente, apresentam graus mais avançados da doença cárie e periodontal pela inadequada higiene oral e difícil manejo interferindo na saúde bucal. Objetivo: verificar o Índice Necessidade de Tratamento Odontológico (INTO) em indivíduos com TEA do município de Passo Fundo – RS. Método: a amostra é composta por 22 indivíduos de ambos os sexos com idades entre 6 e 14 anos, e diferentes graus de TEA. Resultados: 45% das crianças analisadas na pesquisa não necessitavam de tratamento odontológico, 40% tinham de 1 a 3 dentes com necessidade de tratamento e, 13% das crianças tinham de 4 a 8 dentes com necessidade de tratamento odontológico. Conclusão: as meninas apresentam uma saúde bucal geral melhor que os meninos, porém, quando necessitam de tratamento, as necessidades são mais complexas e em maior número; foram poucas as crianças com 4 a 8 dentes necessitando de tratamento odontológico, porém somadas com as crianças com 1 a 3 dentes necessitando de intervenção o número passa da metade das crianças do estudo (54%), ou seja, neste estudo mais da metade das crianças com TEA necessitam de alguma intervenção odontológica

Variants in ATP5F1B are associated with dominantly inherited dystonia

Nasca et al. identify a new candidate gene for dystonia, ATP5F1B, encoding a subunit of the mitochondrial ATP synthase (complex V). Likely pathogenic variants in ATP5F1B were associated with early-onset isolated dystonia in two independent families, both with an autosomal dominant mode of inheritance and incomplete penetrance. ATP5F1B is a subunit of the mitochondrial ATP synthase or complex V of the mitochondrial respiratory chain. Pathogenic variants in nuclear genes encoding assembly factors or structural subunits are associated with complex V deficiency, typically characterized by autosomal recessive inheritance and multisystem phenotypes. Movement disorders have been described in a subset of cases carrying autosomal dominant variants in structural subunits genes ATP5F1A and ATP5MC3. Here, we report the identification of two different ATP5F1B missense variants (c.1000A>C;p.Thr334Pro and c.1445T>C;p.Val482Ala) segregating with early-onset isolated dystonia in two families, both with autosomal dominant mode of inheritance and incomplete penetrance. Functional studies in mutant fibroblasts revealed no decrease of ATP5F1B protein amount but severe reduction of complex V activity and impaired mitochondrial membrane potential, suggesting a dominant-negative effect. In conclusion, our study describes a new candidate gene associated with isolated dystonia and confirms that heterozygous variants in genes encoding subunits of the mitochondrial ATP synthase may cause autosomal dominant isolated dystonia with incomplete penetrance, likely through a dominant-negative mechanism

Building in China - Study trip of the faculty of Civil Engineering of the HTWG Konstanz 2008

Im März 2008 führte die Fakultät Bauingenieurwesen der HTWG Konstanz eine studentische Exkursion nach China durch. Auf dem Programm standen interessante Baustellen Shanghai, Nanjing, Zhenjiang und Beijing sowie der Besuch von Hochschulen. Der Exkursionsbericht beschreibt die besuchten Bauvorhaben und gibt persönliche Eindrücke der Exkursionsteilnehmer wieder.In March 2008 the faculty of civil engineering of the University of Applied Sciences Konstanz, Germany, conducted a study trip for students of civil engineering to China. Construction sites and universities in Shanghai, Nanjing, Zhenjiang and Beijing have been visited. The report describes the places seen and reflects the personal impressions of the participants