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Predictive Modelling with Machine Learning in Plant Breeding
Full text linkGenomic prediction, originally proposed as a solution to the limitations of marker-assisted selection for complex traits, has become the standard for estimating breeding values in both inbred and hybrid crops. While linear models such as GBLUP and RR-BLUP remain effective in many cases, especially when assuming an additive genetic architecture, recent years have seen a growing interest in applying machine learning (ML) methods to overcome some of their constraints, including their limited capacity to model non-additive effects and nonlinear interactions. This thesis explored the influence of three key aspects on the success of genomic prediction: The choice of input features, the statistical model used, and the target trait or crop.
In terms of input features, marker data was compared to minimalist parentage-based models, haplotype blocks, and features generated using autoencoders. It was shown that even simple ML models using parentage-based information can rival marker-based GBLUP under certain conditions, which holds potential for small breeding programs with large amounts of historical, but ungenotyped, records. At the same time, dimensionality reduction techniques, especially a novel haplotype-based autoencoder that was developed during this thesis, were introduced to compress genomic data while preserving prediction accuracy and successfully accelerated model training.
Concerning the model aspect, a variety of ML algorithms were benchmarked using different approaches for hyperparameter tuning. Although no single model outperformed others across all traits and crops, ensemble approaches typically performed better than the individual models they were based on. Support vector machines seemed to be relatively unstable when compared to other ML based algorithms, such as tree-based models.
Finally, results showed that the accuracy of the genomic predictions was strongly dependent on differences between traits, crops with different breeding schemes, and different populations. For hybrids, ML performed well when SCA was more important for determining the hybrid yield than GCA. Large differences were observed for different fungal diseases in wheat, while differences among methods for the same disease were relatively similar.
While ML has not yet provided a significant improvement over traditional methods in many scenarios, its flexibility and potential for multi-modal data integration remain promising. The development of plant breeding-specific model architectures, such as haplotype-based autoencoders, may represent a more promising path than the general application of standard ML models.Sonstige Drittmittelgeber/-inne
Signalment, clinicopathological findings, management practices and comorbidities in cats with diabetes mellitus in Germany: cross-sectional study of 144 cases
Full text linkObjectives: The aim of this study was to describe signalment, clinicopathological findings, management practices and the occurrence of comorbidities in feline diabetes mellitus (DM) in Germany.
Methods: This was a cross-sectional study using questionnaires and laboratory submissions to a commercial laboratory, Antech Lab Germany, between May 2021 and July 2022. Inclusion criteria were diagnosis of DM by the attending veterinarian and submission of a completed questionnaire besides blood samples. Laboratory testing included haematology, serum biochemistry, concentration of total thyroxine (TT4), insulin-like growth factor 1 (IGF-1), cobalamin (COB), fructosamine, b-hydroxybutyrate and DGGR (1,2-O-dilauryl-rac-glycero-3-glutaric acid-[6′-methylresorufin] ester) lipase activity. Data are presented as the median (range) and analysed by non-parametric tests. P <0.05 was considered statistically significant.
Results: The median (range) age of the 144 diabetic cats at diagnosis was 11 years (0.9–18.7), 66.4% were male, 84.6% were domestic shorthair, 50.4% were currently overweight and 61.5% were previously overweight (body condition score >5/9). Most cats were treated with insulin (84%), most commonly protamine zinc insulin (57.5%). Blood glucose curves or continuous glucose monitoring alone or in combination with other methods were performed to adjust insulin therapy in 70.6% of cats. Based on questionnaires, 78.6% were poorly controlled and 21.4% were well controlled. Increased TT4 occurred in 3/139 and hyperthyroidism was known in 5/139 cats (frequency of known/suspected hyperthyroidism: 5.8% [n = 8/139]); 17.5% (n = 17/97) had increased IGF-1 (IGF-1 >746 ng/ml, cut-off for hypersomatotropism with the chemiluminescence assay used in this study); 24.5% (n = 34/139) had COB 746 ng/ml were receiving a higher insulin dose than cats with IGF-1 ≤746 ng/ml (median 1.63 vs 0.86 U/kg/day, P = 0.018).
Conclusion and relevance: Increased DGGR and increased IGF-1 indicating hypersomatotropism are common in diabetic cats and should be tested for. Almost one-quarter of diabetic cats might require COB supplementation
Iron regulatory protein 1-deficient mice exhibit hypospermatogenesis
Full text linkImbalances in testicular iron levels are linked to compromised sperm production and male infertility. Iron regulatory proteins (IRP) 1 and 2 play crucial roles in cellular iron regulation. We investigated the role of IRP1 on spermatogenesis using Irp1-deficient mice (Irp1−/−). Histological analysis of the testis of Irp1−/− mice revealed hypospermatogenesis with a significant reduction in the number of elongated spermatids and daily sperm production compared to wild-type (WT) mice. Flow cytometry of germ cells from WT and Irp1−/− mice showed reduction in spermatocytes and round and elongated spermatids in Irp1−/− mice, which was confirmed by histological and immunofluorescence quantification. Finally, stage VIII of spermatogenesis, crucial for spermatid maturation, was less frequent in Irp1−/− testicular cross-sections. Hypospermatogenesis worsened with age despite unchanged intratesticular iron levels. Mechanistically, this was due to increased oxidative stress indicated by elevated 8-Oxoguanine (8-OxoG) levels, a DNA lesion resulting from reactive oxygen species (ROS). Furthermore, bulk RNA-seq data indicated compromised DNA damage repair and cell cycle processes, including mitosis and meiosis in Irp1−/− mice, which may explain hypospermatogenesis. Our results suggest that IRP1 deletion leads to hypospermatogenesis due to impaired cell cycle progression, decreased DNA damage repair capacity, and oxidative damage. Altogether, this study uncovers a role for IRP1, independent of traditional mechanisms of iron regulation.Deutsche Forschungsgemeinschaft (DFG); ROR-ID:018mejw6
Entschlüsselung von Selbstassemblierungsmechanismen und Chemischen Reaktionen Organischer Moleküle auf Metallsubstraten mittels Chemical Bond Imaging
Full text linkIn recent years, the novel field of on-surface synthesis has been established as one of the main tools for constructing customized, low-dimensional organic nanostructures via bottom-up approaches on atomically flat metal substrates. The self-assembly of the molecular precursors, a process that often serves as pre-step of the on-surface reaction, determines the precise local arrangement of atoms and bonds in neighboring molecules, thus playing a decisive role in product formation. Thereby, intermolecular interactions between hydrogen and fluorine atoms have proven to be a valuable tool to steer molecular alignments. In this work, the mainly unexplored intermolecular hydrogen-fluorine interaction is systematically investigated on inert Au(111) and reactive Cu(111) substrates, using a linear, unilaterally fluorinated 1,2,10,11,12,14-hexafluoropentacene molecule as a model system. In the combined scanning tunneling microscopy and chemical bond imaging study, the local arrangement of hydrogen and fluorine atoms in neighboring molecules is determined in the picometer range and angular variations of a few degrees. While on Au(111) the intermolecular interactions between the molecules are the main contributor to the self-assembly, the higher reactivity of Cu(111) results in different adsorption geometries and molecular arrangements. The highly precise self-assembly study provides new insights into the on-surface interaction of hydrogen and fluorine atoms, thereby highlighting its significance for the field of on-surface synthesis. An on-surface reaction that solely takes place using halogenated precursors is the on-surface Ullmann coupling reaction, which enables the formation of covalent carbon-carbon bonds with the underlying metal surface as a catalyst. However, when applying Ullmann coupling reaction steps, complex self-assembly mechanisms can occur when halogenated precursors interact with the metal surface. Further, cleaved halogens adsorbed on the surface may potentially inhibit the reaction steps. Hence, there is a growing demand for halogen-free precursors for on-surface reactions. Recently, it has been demonstrated that halogen-free (6)Cycloparaphenylene ((6)CPP) molecules are suited to thermally induce a ring-opening polymerization reaction for the synthesis of graphene and biphenylene nanoribbons of confined widths. However, the mechanism of the ring-opening polymerization and the use of cycloparaphenylenes as precursors have not been investigated sufficiently. To contribute to the fundamental understanding of this remarkable reaction, in this thesis we systematically investigate the influence of the ring strain, which decreases with increasing ring size, using a set of cycloparaphenylenes of different sizes ((6)CPP vs. (8)CPP vs. (10)CPP). Our results demonstrate that the ring-opening polymerization is facilitated when using smaller, highly strained cycloparaphenylenes. For larger molecules with lower strain energies, the initial ring-opening is hampered, leading to only partial polymerization for (8)CPP and no polymerization in case of (10)CPP. Additionally, dehydrogenation of individual phenyl rings in intact molecules is observed for (8)CPP and (10)CPP, which further impedes the polymerization reaction.Bundeslände
Midterm Outcomes and Surgical Approaches for Osteochondral Lesions of the Talus
Full text linkBackground: Osteochondral lesions (OCL) of the talus are a significant cause of chronic ankle pain and functional impairment, typically following trauma. Despite advancements in diagnostic imaging and surgical interventions, long-term outcomes vary, and no gold standard treatment has been established.
Methods: This retrospective study evaluated the outcomes of n=64 patients undergoing OCL-related surgery. Inclusion criteria: an OCL of the talus, patients without OCL, with osteoarthritis, or infection were excluded. The cohort was categorized primarily based on the stage of OCL and the surgical technique used: bone marrow stimulation by retrograde or anterograde drilling and microfracturing, transplantation of autologous cancellous bone, and acellular cartilage replacement and other procedures. Postoperative outcomes were assessed using the visual analog scale (VAS), the American Orthopaedic Foot & Ankle Society (AOFAS) score, and the Foot and Ankle Outcome Score (FAOS). Statistical significance was determined using χ2 tests, with P <.05 considered significant.
Results: A total of 97 operations were performed on 64 patients. Fifty-six percent of patients reported complete resolution of symptoms, 25% experienced partial improvement, and 19% showed no improvement. Patients aged ≤30 years had a higher success rate (62.5%) compared with older patients (45.8%, P = .227). Success rates were significantly better for patients with stage II and III lesions (50% and 59%, respectively) than for those with stage IV lesions (P = .043). Material substitution had a success rate of 44.4%, bone marrow stimulation 37.8%, and other procedures 18.8%.
Conclusion: Surgical techniques for treating OCL of the talus provide moderate success, but a significant proportion of patients, especially those with advanced lesions, remain symptomatic
The role of small open reading frames in Shewanella oneidensis phage λSo in host takeover and phage proliferation
Full text linkBacteriophages are the most abundant biological entities on Earth. They wield an immense influence on microbial ecosystems in almost all habitats by regulating bacterial population dynamics. Most phages follow one of two well-characterised strategies for host exploitation: the lytic or the lysogenic cycle. In both pathways, host cell lysis represents the terminal event and is therefore central to phage fitness.
The temperate phage λSo is one of four known prophages in the genome of Shewanella oneidensis MR-1 and has a genome size of about 51 kbp. During lysogeny, λSo remains integrated into the host chromosome, replicating in concert with the host cell. In this study, the lysis system of λSo was characterised as a pinholin-SAR endolysin-two-component spanin pathway. The λSo holin protein, SSo, contains two transmembrane do-mains and also produces an antagonistic isoform through an alternative translation start, named antiholin. This regulatory mechanism enables precise temporal control over the in-itiation of host lysis. In addition to the pinholin and the SAR endolysin, the lysis system requires a two-component spanin complex, made up of an inner membrane protein (i-Spanin, RzSo) and an outer membrane protein (o-Spanin, Rz1So). The corresponding genes are present in an overlapping reading frame structure, and the encoded proteins likely form a functional dimer of two dimers. This putative dimer enables the fusion of the inner and outer membrane. In addition, this work has shown that further, previously uncharacterised gene products are involved in cell lysis.
Like many phages, λSo harbours genes encoding small proteins of unknown function. A gene cluster, so called cluster C, was identified, whose deletion significantly reduced the number of plaque-forming units. Cluster C consists of six genes (lcc1 - lcc6) encoding proteins between 41 and 137 amino acids in length that have no obvious homologies to known protein domains. Bioinformatic analysis suggests that Lcc4 and Lcc6 contain putative transmembrane domains. Functional characterisation revealed that Lcc6 plays a critical role in phage-induced host cell lysis. In lcc6 deletion strains, induction of the lytic cycle of λSo using mitomycin C resulted in the formation of phage particles, which, however, failed to lyse the host cells and are therefore not released. These findings suggest that Lcc6 participates in an early phase of the lysis cascade, likely acting in concert with pinholin-mediated membrane disruption.
The ectopic expression of the Lcc4 protein on the other hand resulted in a pronounced elongation of the host cells and delocalisation of the FtsZ rings - a phenotype that is compatible with a disruption of cell division. The modelling of plausible protein interactions confirmed that this phenotype results from a direct interaction of Lcc4 with key components of the bacterial divisome, particularly FtsZ and ZipA. Site-directed mutagenesis identified isoleucine residues at positions 16 and 19 as essential for the interaction with FtsZ, and tryptophan 80 and arginine 84 as critical for binding to ZipA. Taken together, these results suggest that Lcc4 specifically inhibits bacterial cytokinesis following prophage induction in order to maximise the availability of the metabolic resources of the host cell during phage replication.
The Lcc proteins, encoded by genes of the cluster C, thus represents a previously undescribed phage-host effector system with profound influence on cellular organisation and the course of lysis
Restoring semi-natural grasslands in Central Europe with plant material transfer – achievements, success factors, and knowledge transfer
Full text linkSince the industrial revolution, land use changes and intensification of use have drastically reduced the area of species-rich, semi-natural grasslands in Central Europe. Active restoration of these ecosystems is therefore necessary and increasingly demanded by legislation. One method to (re-)introduce target plant species is the transfer of seed-containing plant material cut from species-rich, semi-natural donor sites. Given the urgency of the restoration task, the aim of this thesis is to contribute to understanding the success factors of this method, particularly in practice.
In a first study, 20 recipient sites of floodplain meadow restoration in Hesse, Germany, were revisited and investigated for vegetation composition in comparison to their corresponding donor sites 13-16 years after plant material transfer. To assess the potential for livestock feeding, biomass yield and energy contents were measured, too. In a second study, 41 recipient-donor site pairs from practical grassland restoration projects in different regions of Germany and Luxembourg spanning a broad moisture gradient were investigated. Vegetation was recorded, and soil analyses and information on restoration from the practice partners were used for success factor identification. In a third study, 33 practitioners of grassland restoration with plant material transfer were interviewed to compare their views on the topic to European scientific literature.
All three studies underline the critical importance of abiotic site conditions supporting the target plant communities. A frequent long-term problem is too high productivity of recipient sites, particularly in floodplain meadows. Site conditions, along with competition for the introduced plant species, are strongly dependent on the previous state of recipient sites. Raw soils, e.g., obtained by topsoil removal, provide low-competitive, nutrient-poor conditions, often enhancing restoration success. Generally, competition for the introduced species is lowered by adequate soil preparation, whose effect may, however, diminish over time. Harvest time for the plant material is decisive for the pool of transferable target species, and supplementing this pool by additional introduction methods, such as sowing, is useful. For maintenance of restored grassland communities, appropriate low-intensive post-restoration management is necessary. Sufficient biomass yield and energy content enable integration in feeding rations for livestock, buffering the costs of management. Practitioner interviews revealed that overarching factors such as project organisation, trust-building, and experience strongly influence how well the previously mentioned conditions can be met. To support grassland restoration in the coming decades, a challenge increased by climate change, intense knowledge exchange between science and practice and among practitioners is advised.Sonstige Drittmittelgeber/-inne
Evaluation of six phosphorus extraction methods for compliance testing of recycled P fertilizers
Full text linkPhosphorus (P) recycling for fertilizer production addresses the dependency on phosphate rock and mitigates P losses to the environment. However, predicting plant-available P in recycled fertilizers is challenging due to their diverse chemical composition. This study aimed at identifying the most suitable P extraction method for fertilizer compliance testing, considering their correlation with actual fertilization efficiency, as well as their simplicity, throughput, recognition and cost. Studies on fertilizer P compliance testing often lack recommendations on minimum P extractability threshold values. Here, thresholds are calculated based on actual fertilization efficiency of a large, chemically diverse set of recycled P fertilizers, many of which are already marketed. Thirty recycled P fertilizers were extracted with H2O, neutral ammonium citrate (NAC), electro-ultrafiltration (EUF), ferrihydrite-filled membranes (iron bag; IB), sodium bicarbonate (NaHCO3) and diffusive gradients in thin films (DGT). The mineral replacement value (MRV) of the fertilizer set was previously evaluated in three pot experiments at a fertilization rate of 50 mg kg−1 soil. MRV correlations with the extractions methods showed similar results for all besides H2O, which cannot be a reliable indicator for P availability. Fertilizers were classified as efficient or inefficient based on their MRV exceeding or falling below 60 % of the triple superphosphate reference value. The minimum P extractability threshold value (MPETV) for each method was based on the efficiency classification and it minimized the number of misclassified fertilizers. NAC, with a 60 % extractable minimum P threshold value, was the most adequate method for compliance testing, despite its overestimation of iron phosphate availability
The Effects of DNA Repair Pathway Engineering on CRISPR-Mediated Genome Editing in Neuronal Cells
Full text linkInherited retinal dystrophies are a group of genetically and clinically heterogeneous disorders that vary in their clinical presentation and progression, possibly leading to blindness. So far, about 332 genes, most expressed in photoreceptors, have been identified to be associated with these diseases. The relatively new genome editing field, particularly CRISPR-Cas9 mediated genome editing technology, which introduces DNA double-strand breaks (DSBs) that are subsequently repaired by the cells' repair systems, has recently drawn much attention. The major repair pathways are non-homologous end-joining (NHEJ) and homology-directed repair (HDR). One of the main obstacles to its application in the retina is the limited knowledge of DNA repair in photoreceptors.
This work aims to analyze the DNA repair mechanisms, improve CRISPR Cas9 genome editing efficacy by modulating and engineering the DNA repair pathways in mature neurons using the human inducible Neurogenin iPS (iNGN) cell line, and validate it as an in vitro model system. These cells are human-induced pluripotent stem cells that differentiate into mature neurons within 4 days. In addition, the iNGN TET3KO cell line was also investigated to determine the impact of the TET3 protein at various differentiation stages.
To achieve this, the iNGN cell lines and a control cell line, HEK293T cells, were transfected with BRET reporter assay plasmid using Cas9 and the inducible Cas systems (iCas). Promoter optimizations were done by replacing the CMV promoter, which can be silenced and suppressed in certain cell types, with a sustained EF1α promoter. Applying the Cas9 system, iNGN WT and iNGN TET3KO cells were tested throughout the differentiation process. Moreover, the modifications of PARP1 protein levels using PARP1 overexpression or knockout plasmids were necessary to study its influence on the DNA repair pathways of different cell lines. The BRET reporter assay was the primary quantitative technique used.
The results showed decreased frameshift rates for the undifferentiated iNGN using the iCas system compared to the Cas9 system, and along with failure to use its timing control advantage, the Cas9 system was mainly used for the remaining experiments. The undifferentiated iNGN TET3KO cells' frameshift rates, as well as at the beginning of differentiation, were significantly higher than iNGN WT ones. Furthermore, the frameshift rate results of the TET3KO overexpressed cells resembled those of iNGN WT cells. After PARP1 modulation, the frameshift rates of PARP1 downregulation were greater throughout the differentiation process of the iNGNs, regardless of the HDR template addition. Interestingly, the results of the iNGN TET3KO cells were higher than those of the WT cells. For future assessment, the generation and application of iNGNs TLR3 cell lines is essential to verify the results obtained using the BRET reporter assay at the genomic level