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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons, resulting in progressive weakness of all voluntary muscles and eventual respiratory failure. Non-motor symptoms, such as cognitive and behavioral changes, frequently occur over the course of the disease. Considering its poor prognosis with a median survival time of 2 to 4 years and limited causal treatment options, an early diagnosis of ALS plays an essential role. In the past, diagnosis has primarily been determined by clinical findings supported by electrophysiological and laboratory measurements. To increase diagnostic accuracy, reduce diagnostic delay, optimize stratification in clinical trials and provide quantitative monitoring of disease progression and treatment responsivity, research on disease-specific and feasible fluid biomarkers, such as neurofilaments, has been intensely pursued. Advances in imaging techniques have additionally yielded diagnostic benefits. Growing perception and greater availability of genetic testing facilitate early identification of pathogenic ALS-related gene mutations, predictive testing and access to novel therapeutic agents in clinical trials addressing disease-modified therapies before the advent of the first clinical symptoms. Lately, personalized survival prediction models have been proposed to offer a more detailed disclosure of the prognosis for the patient. In this review, the established procedures and future directions in the diagnostics of ALS are summarized to serve as a practical guideline and to improve the diagnostic pathway of this burdensome disease.

Increasing fruit yield and quality of tomatoes under organic low-input conditions remains a challenge for producers and breeders. Therefore, it is necessary to identify superior tomato cultivars that are suitable for production and use as parents in breeding programmes. In the present study, the variations in plant morphology and fruit quality characteristics of tomato cultivars were assessed to reveal the traits associated with improved yield and fruit quality. Sixty diverse tomato cultivars were screened in 2015, and in 2016, a subset of 20 cultivars was selected for further evaluation under organic low-input conditions. The results showed high variability among cultivars in all 28 traits that were observed. Salad cultivars had lower plant growth and fruit quality (minerals, dry matter, total soluble solids, and total phenolics) by 10–70%, but they displayed 10–60% higher fruit yield and leaf minerals than cocktail cultivars. Salad tomato cultivars with superior yield and harvest index were mainly derived from breeding for intensive indoor production. Cocktail cultivars with superior yield were mainly derived from organic and outdoor breeding programs. There was a trade-off between fruit yield and quality, indicating a challenge for simultaneous improvement of yield and quality. The importance of Mg was highlighted because of its contribution to the fruit mineral concentration and fruit quality. Cultivars superior in one trait or trait combination under organic low-input conditions were identified to be used by producers and breeders as superior cultivars to meet their production targets and breeding objectives. The importance of Mg provides a novel path for further research on improving soil-available Mg in organic tomato production to enhance fruit mineral concentration and fruit quality in general.

Sensory properties are an essential quality aspect when the consumption of fresh tomato is under consideration. The flavor of tomato is defined as a combination of taste sensations (sweetness, sourness), aroma (volatile compounds), and texture (firmness, mealiness), some of which are proven to be affected by insufficient nutrient supply ��� especially potassium (K). This study intends to undertake a holistic assessment of the K fertilization effect on the flavor of tomato by connecting the use of sensorial and instrumental methods. An optimal K supply significantly increased the sensory descriptors sweetness, sourness, and aroma as well as the instrumental estimated color, firmness, total soluble solids (TSS), titratable acids (TA), and dry matter (DM) in a cultivar-specific manner. The volatile organic compounds (VOCs) were not significantly affected by K fertilization. The evaluation by the panelists confirmed the results of the instru-mental analyses, by which an increment in the fruit quality with rising K supply could be detected. An optimal K supply of 3.66 g/plant could be suggested to increase tomato flavor in the cocktail cultivars studied: Primavera and Yellow Submarine. Cultivar effects should, therefore, be considered for defining the optimal K fertilizer dose that favors high tomato fruit quality and, hence, better flavor., Journal of Applied Botany and Food Quality, Vol. 94 (2021): Journal of Applied Botany and Food Quality

Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuEPO) towards a thermoresistant variant microbial transglutaminase (mTGase), TG16 with the aim of developing novel rHuEPO conjugates that may potentially enhance its biological efficacy. As a model bioconjugation, we studied the reactivity of rHuEPO towards TG16 with a low molar mass amine group containing substrate, monodansyl cadaverine (MDC). The reactions were carried out at a Tm of 54.3 °C, the transition temperature of rHuEPO. Characterization by SDS-PAGE and mass spectrometry confirmed the conjugates formation. Then, we examined the conjugation of rHuEPO with a biodegradable and biocompatible polyester, poly(D-sorbitol adipate) (PDSA). To achieve this, PDSA was enzymatically synthesized using lipase B from Candida antartica (CAL-B), chemically modified with side chains having free primary amine (NH2) groups that can be acyl acceptor substrate of TG16, thoroughly characterized by 1H NMR spectroscopy, and then applied for the TG16-mediated conjugation reaction with rHuEPO. rHuEPO conjugates generated by this approach were identified by SDS-PAGE proving that the amine-grafted PDSA is accepted as a substrate for TG16. The successful conjugation was further verified by the detection of high molar mass fluorescent bands after labelling of amine-grafted PDSA with rhodamine B-isothiocyanate. Overall, this enzymatic procedure is considered as an effective approach to prepare biodegradable rHuEPO-polymer conjugates even in the presence of N- and O-glycans.

The yield and fruit quality parameters of tomatoes are influenced by environmental conditions, and cultivation systems play an important role in improving quality, apart from breeding. We examined five breeding lines and one cultivar in five cultivation systems for yield and fruit quality parameters. The cultivation systems include a single-glazed greenhouse with and without supplementary LED interlighting, a double-glazed greenhouse with and without supplementary LED interlighting, and an organic cultivation system on the field with a rainout shelter. Plants and fruits grown in the double glazing system showed significantly lower values for plant height, yield, DM, TSS, fructose, glucose, antioxidant capacity (DPPH, TEAC), TPC, calcium, phosphorus, and manganese content than in the single-glazed greenhouse, which can be explained by the lower light transmittance. However, it could be seen that the additional LED interlighting could lower the negative effect on yield and quality traits due to double glazing.

Background: The submitted cumulative dissertation is based on two intertwined main studies with biomolecular foundation and clinical perspective on FUS-ALS complemented by two follow-up projects. This subtype of Amyotrophic lateral sclerosis is caused by heterozygous mutations mainly in the NLS of the FUS gene, which interferes with the proper nuclear import of the gene product. To date, there is no sufficient therapy available for this devastating neurodegenerative disease due to an incomplete pathophysiological understanding. Furthermore, not much is known about the specific clinical phenotype of FUS-ALS patients, including the influence of distinct FUS mutations due to the rarity of the disease. FUS is a DNA/RNA-binding protein that is mainly located in the nucleus and has essential functions in splicing, mRNA transport, transcription, and DNA damage repair. Hypothesis:1. It was hypothesized that the human-induced pluripotent stem-cell technique enables to create a sufficient motor neuron in-vitro cell model, which should provide new insights into unknown pathophysiological processes compared to previous cell models of FUS-ALS due to its patient-specific and human character. Thus, screening for potential therapeutic substances should be feasible using this model system. 2. Judging from the previously demonstrated, essential function of FUS in the DNA damage repair, FUS mutations are expected to increase the risk of malignant diseases in affected patients. Moreover, specific correlations between the nature of the mutation and the clinical, neurological phenotype appear plausible.Material & methods: First, an in-vitro cell culture model of FUS-ALS was established. For this purpose, a patient-specific, induced pluripotent stem cell-derived sMN culture was generated, which contained spinal motor neurons with mutations in the gene FUS or WT control cells. The Microfluidic Chamber system was used for the selective analysis of axons, which enabled the live-cell imaging of lysosomes and mitochondria using TIRF microscopy. For the analysis of DNA damage and its repair, gamma-H2A.X immunofluorescence staining was used on the one hand and live-cell laser ablation microscopy on the other, which allowed the precise induction of DNA damage and the monitoring of the repair response. For this purpose, isogenic FUS-GFP cell lines generated via CRISPR-Cas9n were used. A multicentre, retrospective cross-sectional study was conducted to determine genotype-phenotype correlations and the prevalence of malignant neoplasms in FUS-ALS. Previously published FUS-ALS cases have been added to perform a meta-analysis of clinical features.Results: Primarily, correct neuronal differentiation was observed prior to neurodegenerative phenotypes, perfectly mimicking a neurodegenerative disease in the dish. The typical cellular pathology of cytoplasmatic FUS deposition could be reproduced, making it a suitable model for more in-depth pathophysiological studies. Furthermore, the use of Microfluidic Chambers enabled the guided cultivation of neurons with somato-axonal direction of neurite outgrow along tiny microchannels in silico, resulting in a pure motoneuronal, axonal model. Within the distal axonal compartment of these channels, a loss of motility of both lysosomes and mitochondria was observed in parallel with a loss of the mitochondrial membrane potential, followed by the secondary degeneration of the distal axons of the sMNs with FUS mutation. A pathological increase in nuclear DNA damage has been identified as the cause of the distal-axonal phenotypes. This was due to a reduced nuclear FUS abundance as a result of the FUS-NLS mutation, which impaired proper nuclear import. There was evidence of a vicious cycle in this setting because the loss of the nuclear function of FUS disrupted the proper PAR-dependent DNA damage response, resulting in sustained DNA damage. Moreover, the remaining nuclear FUS was transferred into the cytoplasm upon phosphorylation by DNA-PK in a DNA damage response dependent manner, which is to date a process of unclear biological relevance. However, pharmacological inhibition of either the degradation of the PAR biopolymer or DNA-PK improved the nuclear presence of mutant FUS, restored its function in the DNA damage response, and finally prevented the distal axonal phenotype. Furthermore, the multicentric cohort study included 36 newly diagnosed patients. Only one in 40 patients was diagnosed with a malignant disease. By combining the newly diagnosed patients with previously published cases (186 cases in total), the so far most comprehensive database of FUS-ALS patients has been created. This allowed a thorough genotype-phenotype analysis, which showed a clear correlation between individual FUS mutations and the clinical phenotype. Conclusion: The experimental results indicated a primary nuclear insufficiency of mutated FUS, which is due to an impaired nuclear import and leads to a secondary axonal degeneration and finally to neuronal demise (“Dying-Back”). Potential therapeutic options have been identified, but their applicability and safety must be determined in prospective studies. The hypothesis of a generally increased risk of malignant diseases in the analysed FUS-ALS patient group was rejected. However, the clinical data of the meta-analysis are helpful in the counselling of newly diagnosed FUS-ALS patients, including the decision making of the therapeutic management and clearly add FUS-ALS to the family of diseases characterised by deficient DNA damage repair with purely neurological phenotypes such as AOA1, AOA2, and SCAN1.

Species adulteration is a common problem in the coffee trade. Several attempts have been made to differentiate among species. However, finding an applicable methodology that would consider the various aspects of adulteration remains a challenge. This study investigated an ultraviolet&ndash, visible (UV-Vis) spectroscopy-based determination of caffeine and chlorogenic acid contents, as well as the applicability of non-targeted near-infrared (NIR) spectroscopy, to discriminate between green coffee beans of the Coffea arabica (Arabica) and Coffea canephora (Robusta) species from Java Island, Indonesia. The discrimination was conducted by measuring the caffeine and chlorogenic acid content in the beans using UV-Vis spectroscopy. The data related to both compounds was processed using linear discriminant analysis (LDA). Information about the diffuse reflectance (log 1/R) spectra of intact beans was determined by NIR spectroscopy and analyzed using multivariate analysis. UV-Vis spectroscopy attained an accuracy of 97% in comparison to NIR spectroscopy&rsquo, s accuracy by selected wavelengths of LDA (95%). The study suggests that both methods are applicable to discriminate reliably among species.