Das LEADER+ Programm als Motor einer endogenen Regionalentwicklung - Oberallgäu und Vorarlberg im Vergleich

Dieser Beitrag beschäftigt sich mit einer qualitativen Evaluation des LEADER+ Programms zur Entwicklung ländlicher Räume in den Regionen Oberallgäu und Vorarlberg. Anhand der vom LEADER-Konzept geforderten Kriterien werden die Ergebnisse von Expertengesprächen dargelegt, die merklichen Entwicklungsunterschiede zwischen den beiden benachbarten Regionen aufgezeigt, Entwicklungshemmnisse, Erfolgsfaktoren und Handlungsempfehlungen abgeleitet. Deutlich wird u. a., dass die längere Projektlaufzeit in Vorarlberg und der reichere Erfahrungsschatz zu den besser entwickelten Wertschöpfungsketten und der intensiveren regionalen Zusammenarbeit der Akteure beitragen. Insgesamt ist LEADER+ als sinnvolle projektbezogene Ergänzungsmaßnahme zur klassischen Direktförderung der Landwirtschaft anzusehen, da sektoren- und branchenübergreifende Projekte gefördert werden, die Eigeninitiative und regionale Kooperation der Betroffenen erfordern. Damit kann ein Beitrag zur Überwindung des kommunalen „Kirchturmdenkens“ geleistet werden. Regionale Identität und das Problembewusstsein der Bevölkerung werden geschärft und interregionale Kooperationen werden angestoßen.This paper presents a qualitative evaluation of the LEADER+ programme to support the development of rural space in the Upper Allgäu and Vorarlberg regions. Applying the criteria demanded under the LEADER concept, the paper sets out the results of expert interviews, describes the clear differences in development between these two neighbouring regions, and on this basis identifies both impediments to development and success factors, and derives recommendations for action. One point which emerges is that the longer duration of the project in Vorarlberg, combined with the greater wealth of experience, together account for more highly developed value-added chains and to generally more intensive regional co-operation among the actors involved. Overall, LEADER+ can be regarded as a sensible project-based supplement to the classical instrument of direct subsidies to agriculture as it supports both cross-sectoral and crossbusiness projects which call for both personal initiative and regional identification on the part of those involved. It thus contributes to overcoming the parochial attitudes sometimes displayed by local authorities. At the same time it promotes both a sense of regional identity and problem awareness and provides an impetus for interregional cooperation

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs

Applications of Micro-Raman Spectroscopy in Cultural Heritage – Examples from the Laboratory for Conservation Research of the Collections Centre of the Swiss National Museums

The paper reports on three applications of micro-Raman spectroscopy which were carried out in the Laboratory for Conservation Research of the Collections Centre of the Swiss National Museums. The first application addresses archaeological questions about three early mediaeval fibulae which were decorated with gemstones. The determination of different corrosion products on pigeon cameras by micro-Raman spectroscopy is described in the second part of this article. The last case study discusses an example from the field of preventive conservation where changes on metal coupons from the so-called Oddy test were analysed

Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients

Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.Peer Reviewe

Tetramer enrichment reveals the presence of phenotypically diverse hepatitis C virus-specific CD8+T cells in chronic infection

Virus-specific CD8+ T cells are rarely detectable ex vivo by conventional methods during chronic hepatitis C virus (HCV) infection. In this study, however, we were able to detect and characterize HCV-specific CD8+ T cells in all chronically HCV genotype 1a-infected, HLA-A*02:01-positive patients analyzed by performing major histocompatibility complex (MHC) class I tetramer enrichment. Two-thirds of these enriched HCV-specific CD8+ T-cell populations displayed an effector memory phenotype, whereas, surprisingly, one-third displayed a naive-like phenotype despite ongoing viral replication. CD8+ T cells with an effector memory phenotype could not expand in vitro, suggesting exhaustion of these cells. Interestingly, some of the naive-like CD8+ T cells proliferated vigorously upon in vitro priming, whereas others did not. These differences were linked to the corresponding viral sequences in the respective patients. Indeed, naive-like CD8+ T cells from patients with the consensus sequence in the corresponding T-cell epitope did not expand in vitro. In contrast, in patients displaying sequence variations, we were able to induce HCV-specific CD8+ T-cell proliferation, which may indicate infection with a variant virus. Collectively, these data reveal the presence of phenotypically and functionally diverse HCV-specific CD8+ T cells at very low frequencies that are detectable in all chronically infected patients despite viral persistence. IMPORTANCE In this study, we analyzed CD8+ T-cell responses specific for HLA-A*02:01-restricted epitopes in chronically HCV-infected patients, using MHC class I tetramer enrichment. Importantly, we could detect HCV-specific CD8+ T-cell populations in all patients. To further characterize these HCV-specific CD8+ T-cell populations that are not detectable using conventional techniques, we performed phenotypic, functional, and viral sequence analyses. These data revealed different mechanisms for CD8+ T-cell failure in HCV infection, including T-cell exhaustion, viral escape, and functional impairment of naive-like HCV-specific CD8+ T cells

Porcine model elucidates function of p53 isoform in carcinogenesis and reveals novel circTP53 RNA

Recent years have seen an increasing number of genetically engineered pig models of human diseases including cancer. We previously generated pigs with a modified TP53 allele that carries a Cre-removable transcriptional stop signal in intron 1, and an oncogenic mutation TP53R167H (orthologous to human TP53R175H) in exon 5. Pigs with the unrecombined mutant allele (flTP53R167H) develop mainly osteosarcoma but also nephroblastomas and lymphomas. This observation suggested that TP53 gene dysfunction is itself the key initiator of bone tumorigenesis, but raises the question which aspects of the TP53 regulation lead to the development of such a narrow tumour spectrum. Molecular analysis of p53 revealed the presence of two internal TP53 promoters (Pint and P2) equivalent to those found in human. Consequently, both pig and human express TP53 isoforms. Data presented here strongly suggest that P2-driven expression of the mutant R167H-Δ152p53 isoform (equivalent to the human R175H-Δ160p53 isoform) and its circular counterpart circTP53 determine the tumour spectrum and play a critical role in the malignant transformation in flTP53R167H pigs. The detection of Δ152p53 isoform mRNA in serum is indicative of tumorigenesis. Furthermore, we showed a tissue-specific p53-dependent deregulation of the p63 and p73 isoforms in these tumours. This study highlights important species-specific differences in the transcriptional regulation of TP53. Considering the similarities of TP53 regulation between pig and human, these observations provide useful pointers for further investigation into isoform function including the novel circTP53 in both the pig model and human patients.ISSN:0950-9232ISSN:1476-559

PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence-free survival in HCC patients

B Cell Numbers Predict Humoral and Cellular Response Upon SARS–CoV-2 Vaccination Among Patients Treated With Rituximab

Objective: Patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) therapy are at higher risk of poor COVID-19 outcomes and show substantially impaired humoral immune response to anti-SARS-CoV-2 vaccine. However, the complex relationship between antigen-specific B cells and T cells and the level of B cell repopulation necessary to achieve anti-vaccine responses remain largely unknown. Methods: Antibody responses to SARS-CoV-2 vaccines and induction of antigen-specific B and CD4/CD8 T cell subsets were studied in 19 patients with rheumatoid arthritis (RA) or antineutrophil cytoplasmic antibody-associated vasculitis receiving RTX, 12 patients with RA receiving other therapies, and 30 healthy controls after SARS-CoV-2 vaccination with either messenger RNA or vector-based vaccines. Results: A minimum of 10 B cells per microliter (0.4% of lymphocytes) in the peripheral circulation appeared to be required for RTX-treated patients to mount seroconversion to anti-S1 IgG upon SARS-CoV-2 vaccination. RTX-treated patients who lacked IgG seroconversion showed reduced receptor-binding domain-positive B cells (P = 0.0005), a lower frequency of Tfh-like cells (P = 0.0481), as well as fewer activated CD4 (P = 0.0036) and CD8 T cells (P = 0.0308) compared to RTX-treated patients who achieved IgG seroconversion. Functionally relevant B cell depletion resulted in impaired interferon-γ secretion by spike-specific CD4 T cells (P = 0.0112, r = 0.5342). In contrast, antigen-specific CD8 T cells were reduced in both RA patients and RTX-treated patients, independently of IgG formation. Conclusion: In RTX-treated patients, a minimum of 10 B cells per microliter in the peripheral circulation is a candidate biomarker for a high likelihood of an appropriate cellular and humoral response after SARS-CoV-2 vaccination. Mechanistically, the data emphasize the crucial role of costimulatory B cell functions for the proper induction of CD4 responses propagating vaccine-specific B cell and plasma cell differentiation

Temporary antimetabolite treatment hold boosts SARS-CoV-2 vaccination–specific humoral and cellular immunity in kidney transplant recipients

Transplant recipients exhibit an impaired protective immunity after SARS-CoV-2 vaccination, potentially caused by mycophenolate (MPA) immunosuppression. Recent data from patients with autoimmune disorders suggest that temporary MPA hold might greatly improve booster vaccination outcomes. We applied a fourth dose of SARS-CoV-2 vaccine to 29 kidney transplant recipients during a temporary (5 weeks) MPA/azathioprine hold, who had not mounted a humoral immune response to previous vaccinations. Seroconversion until day 32 after vaccination was observed in 76% of patients, associated with acquisition of virus-neutralizing capacity. Interestingly, 21/25 (84%) calcineurin inhibitor-treated patients responded, but only 1/4 belatacept-treated patients responded. In line with humoral responses. counts and relative frequencies of spike receptor binding domain-specific (RBD-specific) B cells were markedly increased on day 7 after vaccination, with an increase in RBD-specific CD27(++)CD38(+) plasmablasts. Whereas overall proportions of spike-reactive CD4(+) T cells remained unaltered after the fourth dose, frequencies were positively correlated with specific IgG levels. Importantly, antigen-specific proliferating Ki67(+) and in vivo-activated programmed cell death 1-positive T cells significantly increased after revaccination during MPA hold, whereas cytokine production and memory differentiation remained unaffected. In summary, antimetabolite hold augmented all arms of immunity during booster vaccination. These data suggest further studies of antimetabolite hold in kidney transplant recipients