Optimising Gene Therapy for X-linked Retinitis Pigmentosa

Purpose Mutations in RPGRORF15 cause 70 to 90 % of the monogenetic disease X-linked retinitis pigmentosa (XLRP), making this gene a high-yield target for causal treatment with gene therapy. Due to the purine-rich, repetitive nature of the terminal ORF15 exon, maintaining transgene sequence fidelity has proven to be a road-block in translational efforts. This thesis contributes to the optimisation of a gene therapy for RPGR-XLRP in two ways: firstly, it aims to investigate codon optimization and use of mutant AAV capsids as a means to overcome the inherent instability of RPGRORF15 and increase transgene expression. Secondly, analysis of pre-treatment characteristics in a cohort of 50 RPGR-XLRP patients will assist both future prospective observational and interventional trials by determining symmetry of disease, rate of progression and suitability of outcome measures as endpoints for clinical trials. Methods In the first part of the thesis, Western Blot was used to quantify transgene expression in HEK293T cells transfected with codon optimised (co) or wild type (wt) RPGR plasmids as well as to detect transgene expression in mice unilaterally injected with AAV2/8.coRPGR. Immunolabeling was used to show correct localisation of codon optimised transgene to the photoreceptor cilium and to compare transduction efficiency between wild type and single mutant AAV8Y733F capsids. In the thesis’ second part, a retrospective, cross-sectional analysis of 50 patients extracted visual acuity, visual fields (I4e and III4e targets), foveal thickness and ERG data points (ISCEV standard protocol) alongside molecular genetic data. Symmetry and progression were assessed using linear regression and cross-sectional analysis, respectively. Kaplan-Meyer Curves were used to estimate cumulative ‘survival’ of three important levels of visual function (full vision, reading ability, threshold to legal blindness) with age. Results HEK293T cells transfected with p.coRPGR showed an increase in protein expression (p < 0.005) and demonstrated a superior transgene stability compared to the wild type control. Three different mouse lines, C57BL/6J, C57BL/6J Rd9/Boc and Rpgr-/y, treated with AAV2/8.coRPGR showed a reliable, albeit variable transgene expression and demonstrated co-localisation with RPGR interacting protein (RPGRIP) in the connecting cilium. Mutant capsid (AAV8Y733F) failed to show a significant increase in transduction of 661W cone-like photoreceptor cells (p = 0.058). In the retrospective analysis of clinical data from XLRP patients, 73 % of exonic mutations occurred in ORF15. Yet no clear genotype-phenotype relationship could be established between mutations located in these two parts of the RPGR gene and patients with ORF15 mutations did not have a significantly different visual acuity (p = 0.9) or visual field (III4e; p = 0.6) than those with mutations in exons 1-14. Comparison of both eyes revealed a strong symmetry of degeneration in all outcome measures, with visual fields (I4e ρ = 0.99; III4e ρ = 0.96) and ERG (30 Hz flicker ρ = 0.95) exhibiting the highest symmetry. Disease progression eluded description by a simple function. Kaplan-Meier curve (KMC) analysis predicts the most severe decline in vision between the third and fourth decade of life. Conclusions Codon optimisation of RPGR significantly increased transgene levels in HEK293T cells compared to a wild type RPGR expression cassette. AAV2/8.coRPGR injected mouse eyes reliably expressed RPGR protein that correctly localised to the photoreceptor connecting cilium in mouse models of RPGR-XLRP. High symmetry in all outcome measures confirm that the contralateral eye can be used as an internal control in an RPGR-XLRP gene therapy trial. The variability between patients makes an intra-individual control preferable to an inter-individual control. According to these findings, the most sensitive parameter to measure disease progression and treatment success in an interventional RPGR-XLRP trial seems to be kinetic visual field using the III4e target. Overall, these two pillars of research contribute to the foundation enabling translation of RPGRORF15 gene therapy into a clinical trial

Fostering the Implementation of Nature Conservation Measures in Agricultural Landscapes: The NatApp

Large-scale, high-input, and intensified agriculture poses threats to sustainable agroecosystems and their inherent biodiversity. The EU Common Agricultural Policy (CAP) covers a great number of nature conservation programs (Agri-Environment and Climate Measures, AECM) aiming to encourage sustainable agriculture. Currently, farmers are not encouraged to broadly implement these measures due to the lack of structured information, overly complicated and unclear application procedures, and a high risk of sanctions. In addition, the current structures are associated with time-consuming monitoring and control procedures for the paying agencies. Digital technologies can offer valuable assistance to circumvent relevant barriers and limitations and support a broader uptake of AECM. NatApp is a digital tool that supports and guides farmers through the complete process of choosing, applying, implementing, and documenting AECM on their fields in accordance with legal requirements in Germany. We introduce the concept of NatApp and analyze how it can simplify and encourage the uptake and implementation of AECM. This study identifies its unique features for the provision of information and documentation opportunities compared with other digital farming tools focused on sustainable agriculture and outline how it can support farmers to actively contribute to more sustainable agriculture.“Cross-federal state implementation study on the use of the nature conservation app (NatApp) in agricultural and administrative practice-NatApp 2.0”Peer Reviewe

Potenziale des Sojabohnenanbaus in Nord-Ostdeutschland: Einfluss von Beregnung und Sojasorte auf Ertrag, N2-Fixierung und Vorfruchtwirkung

Sojabohnen sind in weiten Teilen Europas eine Körnerleguminose mit großem ökonomischen Potential. Sojabohnen erzielen unter warmen und ausreichend feuchten Bedingungen relativ hohe Erträge und sind so eine interessante Alternative zu anderen Körnerleguminosen. Unter weniger optimalen Bedingungen, wie sie mit geringen Niederschlägen und sandigen Böden in weiten Teilen Nord-Ostdeutschlands vorliegen, wurden bisher nur unzureichende Untersuchungen zum Potenzial der Sojabohnen durchgeführt. Die Ziele dieser Studie sind daher, (i) standortangepasste Sojasorten zu identifizieren, (ii) den Einfluss von Beregnung auf Ertrag und N2-Fixierung von Sojabohnen zu quantifizieren, (iii) einen Ertragsvergleich zwischen Soja und Blauer Süßlupine durchzuführen und (iv) den Vorfruchtwert von Soja im Vergleich zur Blauen Lupine und Buchweizen zu untersuchen

Statistical Analysis versus the M5P Machine Learning Algorithm to Analyze the Yield of Winter Wheat in a Long-Term Fertilizer Experiment

To compare how different analytical methods explain crop yields from a long-term field experiment (LTFE), we analyzed the grain yield of winter wheat (WW) under different fertilizer applications in Müncheberg, Germany. An analysis of variance (ANOVA), linear mixed-effects model (LMM), and MP5 regression tree model were used to evaluate the grain yield response. All the methods identified fertilizer application and environmental factors as the main variables that explained 80% of the variance in grain yields. Mineral nitrogen fertilizer (NF) application was the major factor that influenced the grain yield in all methods. Farmyard manure slightly influenced the grain yield with no NF application in the ANOVA and M5P regression tree. While sources of environmental factors were unmeasured in the ANOVA test, they were quantified in detail in the LMM and M5P model. The LMM and M5P model identified the cumulative number of freezing days in December as the main climate-based determinant of the grain yield variation. Additionally, the temperature in October, the cumulative number of freezing days in February, the yield of the preceding crop, and the total nitrogen in the soil were determinants of the grain yield in both models. Apart from the common determinants that appeared in both models, the LMM additionally showed precipitation in June and the cumulative number of days in July with temperatures above 30 °C, while the M5P model showed soil organic carbon as an influencing factor of the grain yield. The ANOVA results provide only the main factors affecting the WW yield. The LMM had a better predictive performance compared to the M5P, with smaller root mean square and mean absolute errors. However, they were richer regressors than the ANOVA. The M5P model presented an intuitive visualization of important variables and their critical thresholds, and revealed other variables that were not captured by the LMM model. Hence, the use of different methods can strengthen the statement of the analysis, and thus, the co-use of the LMM and M5P model should be considered, especially in large databases involving multiple variables.Peer Reviewe

Wie tief und intensiv wurzeln Sojabohnen unter kontinental geprägten Bedingungen Zentraleuropas?

Mit Hilfe der Profilwandmethode wird die Wurzellängendichte von Sojabohnen an zwei verschiedenen Standorten in Ostdeutschland untersucht

Plant growth response of broad bean (Vicia faba L.) to biochar amendment of loamy sand soil under irrigated and drought conditions

The broad bean (Vicia faba L.) originated in the Near East, and is cultivated around the world, however, its cultivation is affected by drought stress in several central growing regions of the globe. The present study was designed to determine the effect of biochar on bean plant growth, acquisition of nitrogen (N), phosphorus (P), and potassium (K) and on soil nutrient contents under drought and irrigated conditions. Pyrolysis char from maize (MBC) at 2 and 4% concentrations was used for pot experiments. The shoot and/or root biomass of bean grown in soil amended with 2 and 4% MBC under irrigated condition was increased. Furthermore, increased nodule numbers of bean grown at 4% MBC amendment was observed under both irrigated and drought conditions. P and K uptake of plants under drought conditions increased by 14% and 23% under 2% MBC amendment, and by 23% and 34% under 4% MBC amendment as compared to plants grown without biochar application, respectively. This study demonstrated beneficial effects of biochar produced from maize on growth and nutrient uptake of broad bean, by improving the nodule formation and soil nutritional contents in a sandy loam soil.Peer Reviewe

Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant

Background Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms. Purpose To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing. Methods Case report. Results A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated “read-through” of the premature stop codon, the resulting missense changes were predicted to be “possibly damaging” in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window. Conclusions This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants

Psychosocial Hazard Analysis in a Heterogeneous Workforce: Determinants of Work Stress in Blue- and White-Collar Workers of the European Steel Industry

The European steel industry’s workforce is highly heterogeneous and consists of various occupational groups, presumably facing different psychosocial stressors. The few existing studies on the subject mainly focused on physical constraints of blue-collar workers, whereas the supposable psychosocial workload received only little research attention. This is remarkable considering the challenges associated with statutory required risk assessment of psychosocial hazards. Valid measures of hazard analysis must account for various stressors and reliably identify them, also between occupational groups. The present study, based on a sample of blue- and white-collar workers (N = 124) from the European steel industry, aims to provide a first insight into psychosocial stressors and strain at work in this rarely researched industrial sector. Furthermore, two well-known theoretical roadmaps in job analysis are examined regarding their utility for risk assessment in heterogeneous workforces: the German standard version of the Copenhagen Psychosocial Questionnaire (COPSOQ) and the short version of the effort–reward imbalance questionnaire. Hierarchical multiple regression analyses revealed that the COPSOQ was better suited to predict various strain indices in the present sample. Especially stressors relating to socioemotional aspects, such as work-privacy conflict, revealed a reasonable impact, indicating the need for comprehensive solutions at the organizational level instead of solutions focusing on single workplaces. To conclude, a broadly diversified and validated approach in psychosocial risk assessment is needed to adequately assess the variety of psychosocial factors at work and in different occupational groups

The Distribution and Migration of 137Cs in Oak (Quercus serrata) and Cedar (Cryptomeria japonica) Forest Organic Fractions

To analyse the 137Cs distribution and migration under various fractions of organic matter layers, this study investigated easily recognizable, originally shaped organic L-fractions, and not easily recognizable, early fermented and fragmented organic F-fractions, of both oak (Quercus serrata) and cedar (Cryptomeria japonica) sampled from Osawa watershed sites at Nihonmatsu City, Fukushima Prefecture, Japan. The organic materials were put on top of soil columns from Field Museum (FM) Tamakyuryo in Hachioji City, Tokyo. The 137Cs vertical distribution in forest soil profiles was analyzed using the relaxation mass depth, ho (kg m−2). Soil columns with both L and F- organic layer fractions of both oak and cedar, labelled as Oak-L, Oak-F, Cedar-L and Cedar-F with four replications (n = 16), were set up by the laboratory column-based method and kept under five months’ incubation period. Soil columns after incubation were sampled at depths of 0–1 cm, 1–2 cm, 2–5 cm and 5–10 cm. Results of 137Cs inventory in the organic fractions showed that 86% (oak and cedar) of the total organic layer fractions 137Cs inventory accumulated within the F-layer, indicating that the transformation of litter is a huge source for potentially mobile 137Cs, especially the oak F-layer (67% 137Cs inventory) and further continuous transfer into the forest soil mineral layers. A higher ho in L treated soils (Oak-L and Cedar-L) compared to the F treatments implied that the low 137Cs amounts penetrated faster and deeper due to their water-soluble nature. Furthermore, Cedar-F showed a higher ho of 24.3 kg m−2 than Oak-F of ho, 14.0 kg m−2, and a significant positive relationship between 137Cs retention and total carbon (TC) (p < 0.05) suggested the influence of soil organic matter on 137Cs penetration and retention. The C/N (carbon nitrogen ratio) results revealed that organic matter fractions of high C/N including 137Cs, as observed in Cedar-F, in which decomposition does not advance, penetrates soil depths while the organic matter fraction of low C/N, observed in Oak-F, showed that decomposition advanced to release 137Cs which was held by adsorption unto the RIP (radiocesium interception potential) of soil surface. In addition, infiltration by water as a transportation process was suggested to largely influence the downward migration and retention of 137Cs at lower depths of Cedar-F.Japan Society for the Promotion of SciencePeer Reviewe