Amyotropic lateral sclerosis (ALS) is a lethally progressive and irreversible neurodegenerative disease marked by apparent death of motor neurons present in the spinal cord, brain stem and motor cortex. While more and more gene mutants being established for genetic ALS, the vast majority suffer from sporadic ALS (>90%). It has been challenging, thus, to model sporadic ALS which is one reason why the underlying pathophysiology remains elusive and has stalled the development of therapeutic strategies of this progressive motor neuron disease. To further unravel these pathological signaling pathways, human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs) from FUS- and SOD1 ALS patients and healthy controls were systematically compared to independent published datasets. Here through this study we created a gene profile of ALS by analyzing the DEGs, the Kyoto encyclopedia of Genes and Genomes (KEGG) pathways, the interactome and the transcription factor profiles (TF) that would identify altered molecular/functional signatures and their interactions at both transcriptional (mRNAs) and translational levels (hub proteins and TFs). Our findings suggest that FUS and SOD1 may develop from dysregulation in several unique pathways and herpes simplex virus (HSV) infection was among the topmost predominant cellular pathways connected to FUS and not to SOD1. In contrast, SOD1 is mainly characterized by alterations in the metabolic pathways and alterations in the neuroactive-ligand-receptor interactions. This suggests that different genetic ALS forms are singular diseases rather than part of a common spectrum. This is important for patient stratification clearly pointing towards the need for individualized medicine approaches in ALS.

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder causing progressive loss of motor neurons. Mutations in Fused in sarcoma (FUS) leading to its cytoplasmic mislocalization cause a subset of ALS. Under stress, mutant FUS localizes to stress granules (SGs)-cytoplasmic condensates composed of RNA and various proteins. Aberrant dynamics of SGs is linked to the pathology of ALS. Here, using motor neurons (MNs) derived from human induced pluripotent stem cells, we show that, in mutant FUS, MN dynamics of SGs is disturbed. Additionally, heat-shock response (HSR) and integrated stress response (ISR) involved in the regulation of SGs are upregulated in mutant MNs. HSR activation correlates with the amount of cytoplasmic FUS mislocalization. While inhibition of SG formation, translation, or ISR does not influence survival of FUS ALS neurons, proteotoxicity that cannot be compensated with the activation of stress pathways is the main driver of neurodegeneration in early FUS ALS.

Sulfite reductase (SIR) is a key enzyme in higher plants in the assimilatory sulfate reduction pathway. SIR, being exclusively localized in plastids, catalyzes the reduction of sulfite (SO3 2-) to sulfide (S2-) and is essential for plant life. We characterized transgenic plants leading to co-suppression of the SIR gene in tobacco (Nicotiana tabacum cv. Samsun NN). Co-suppression resulted in reduced but not completely extinguished expression of SIR and in a reduction of SIR activity to about 20-50% of the activity in control plants. The reduction of SIR activity caused chlorotic and necrotic phenotypes in tobacco leaves, but with varying phenotype strength even among clones and increasing from young to old leaves. In transgenic plants compared to control plants, metabolite levels upstream of SIR accumulated, such as sulfite, sulfate and thiosulfate. The levels of downstream metabolites were reduced, such as cysteine, glutathione (GSH) and methionine. This metabolic signature resembles a sulfate deprivation phenotype as corroborated by the fact that O-acetylserine (OAS) accumulated. Further, chlorophyll contents, photosynthetic electron transport, and the contents of carbohydrates such as starch, sucrose, fructose, and glucose were reduced. Amino acid compositions were altered in a complex manner due to the reduction of contents of cysteine, and to some extent methionine. Interestingly, sulfide levels remained constant indicating that sulfide homeostasis is crucial for plant performance and survival. Additionally, this allows concluding that sulfide does not act as a signal in this context to control sulfate uptake and assimilation. The accumulation of upstream compounds hints at detoxification mechanisms and, additionally, a control exerted by the downstream metabolites on the sulfate uptake and assimilation system. Co-suppression lines showed increased sensitivity to additionally imposed stresses probably due to the accumulation of reactive compounds because of insufficient detoxification in combination with reduced GSH levels.

Neurodegenerative diseases pose a complex field with various neuronal subtypes and distinct differentially affected intra-neuronal compartments. Modelling of neurodegeneration requires faithful in vitro separation of axons and dendrites, their distal and proximal compartments as well as organelle tracking with defined retrograde versus anterograde directionality. We use microfluidic chambers to achieve compartmentalization and established high throughput live organelle imaging at standardized distal and proximal axonal readout sites in iPSC-derived spinal motor neuron cultures from human amyotrophic lateral sclerosis patients to study trafficking phenotypes of potential disease relevance. Our semi-automated pipeline of organelle tracking with FIJI and KNIME yields quantitative, multiparametric high content phenotypic signatures of organelle morphology and their trafficking in axons. We provide here the resultant large datasets to enable systemic signature interrogations for comprehensive and predictive disease modelling, mechanistic dissection and secondary hit validation (e.g. drug screens, genetic screens). Due to the nearly complete coverage of analysed motility events, our quantitative method yields a bias-free statistical power superior over common analyses of a handful of manual kymographs.

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient-albeit to a smaller extent-to induce premature distal axonal trafficking deficits and increased DSBs.

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.

The limited availability of phosphorus (P) in soils causes a major constraint in the productivity of potatoes, which requires increased knowledge of plant adaptation responses in this condition. In this study, six potato cultivars, namely, Agria, Lady Claire, Milva, Lilly, Sieglinde, and Verdi, were assessed for their responses on plant growth, leaf physiology, P use efficiency (PUE), and tuber quality with three P levels (Plow, Pmed, and Phigh). The results reveal a significant variation in the cultivars in response to different P availabilities. P-efficient cultivars, Agria, Milva, and Lilly, possessed substantial plant biomass, tuber yield, and high P uptake efficiency (PUpE) under low P supply conditions. The P-inefficient cultivars, Lady Claire, Sieglinde, and Verdi, could not produce tubers under P deprivation conditions, as well as the ability to efficiently uptake P under low-level conditions, but they were efficient in P uptake under high soil P conditions. Improved PUpE is important for plant tolerance with limited P availability, which results in the efficient use of the applied P. At the leaf level, increased accumulations of nitrate, sulfate, sucrose, and proline are necessary for a plant to acclimate to P deficiency-induced stress and to mobilize leaf inorganic phosphate to increase internal PUE and photosynthesis. The reduction in plant biomass and tuber yield under P-deficient conditions could be caused by reduced CO2 assimilation. Furthermore, P deficiency significantly reduced tuber yield, dry matter, and starch concentration in Agria, Milva, and Lilly. However, contents of tuber protein, sugars, and minerals, as well as antioxidant capacity, were enhanced under these conditions in these cultivars. These results highlight the important traits contributing to potato plant tolerance under P-deficient conditions and indicate an opportunity to improve the P efficiency and tuber quality of potatoes under deficient conditions using more efficient cultivars. Future research to evaluate molecular mechanisms related to P and sucrose translocation, and minimize tuber yield reduction under limited P availability conditions is necessary.

Low phosphorus (P) availability is a major limiting factor for potatoes. P fertilizer is applied to enhance P availability; however, it may become toxic when plants accumulate at high concentrations. Therefore, it is necessary to gain more knowledge of the morphological and biochemical processes associated with P deficiency and toxicity for potatoes, as well as to explore an alternative approach to ameliorate the P deficiency condition. A comprehensive study was conducted (I) to assess plant morphology, mineral allocation, and metabolites of potatoes in response to P deficiency and toxicity; and (II) to evaluate the potency of plant growth-promoting rhizobacteria (PGPR) in improving plant biomass, P uptake, and metabolites at low P levels. The results revealed a reduction in plant height and biomass by 60-80% under P deficiency compared to P optimum. P deficiency and toxicity conditions also altered the mineral concentration and allocation in plants due to nutrient imbalance. The stress induced by both P deficiency and toxicity was evident from an accumulation of proline and total free amino acids in young leaves and roots. Furthermore, root metabolite profiling revealed that P deficiency reduced sugars by 50-80% and organic acids by 20-90%, but increased amino acids by 1.5-14.8 times. However, the effect of P toxicity on metabolic changes in roots was less pronounced. Under P deficiency, PGPR significantly improved the root and shoot biomass, total root length, and root surface area by 32-45%. This finding suggests the potency of PGPR inoculation to increase potato plant tolerance under P deficiency.

FUS (fused in sarcoma) plays a key role in several steps of RNA metabolism, and dominant mutations in this protein are associated with neurodegenerative diseases. Here, we show that FUS is a component of the cellular response to topoisomerase I (TOP1)-induced DNA breakage; relocalising to the nucleolus in response to RNA polymerase II (Pol II) stalling at sites of TOP1-induced DNA breaks. This relocalisation is rapid and dynamic, reversing following the removal of TOP1-induced breaks and coinciding with the recovery of global transcription. Importantly, FUS relocalisation following TOP1-induced DNA breakage is associated with increased FUS binding at sites of RNA polymerase I transcription in ribosomal DNA and reduced FUS binding at sites of RNA Pol II transcription, suggesting that FUS relocates from sites of stalled RNA Pol II either to regulate pre-mRNA processing during transcriptional stress or to modulate ribosomal RNA biogenesis. Importantly, FUS-mutant patient fibroblasts are hypersensitive to TOP1-induced DNA breakage, highlighting the possible relevance of these findings to neurodegeneration.

Salmonella enterica causes an estimated 1 million illnesses in the United States each year, resulting in 19,000 hospitalizations and 380 deaths, and is one of the four major global causes of diarrhoeal diseases. No effective treatments are available to the food industry. Much attention has been given to colicins, natural non-antibiotic proteins of the bacteriocin class, to control the related pathogen Escherichia coli. We searched Salmonella genomic databases for colicin analogues and cloned and expressed in plants five such proteins, which we call salmocins. Among those, SalE1a and SalE1b were found to possess broad antimicrobial activity against all 99 major Salmonella pathovars. Each of the two salmocins also showed remarkably high potency (>106 AU/µg recombinant protein, or >103 higher than colicins) against major pathogenic target strains. Treatment of poultry meat matrices contaminated with seven key pathogenic serovars confirmed salmocin efficacy as a food safety intervention against Salmonella.

Consumer complaints about the flavor of fresh tomato fruits (Solanum lycopersicum L.) have increased in the past few decades, and numerous studies have been done on the flavor of tomatoes and how it is influenced. However, it has not yet been taken into account how consumer handling affects the flavor when considering the complete post-harvest chain-from retailer (distributor) to retail to consumer. In this study, the impact of two household storage regimes on the volatile profile and important flavor-related compounds were examined, considering the entire post-harvest handling. New breeding lines (n = 2) and their parental cultivars (n = 3) were evaluated. Fruits were harvested ripe and stored at 12.5°C for 1 day, at 20°C for 2 days, and afterward at either 20 or 7°C for another 4 days. The aroma volatile profile was measured using GC-MS and GC-FID. A trained panel was used to characterize the sensory attributes of the fruits. In both storage regimes, the relative amount of hexanal increased during the storage period in three of the five cultivars/breeding lines while benzaldehyde was the only volatile compound that decreased significantly in four cultivars/breeding lines. The relative concentration of the precursors of lipid-derived volatiles-linoleic (C18:2) and linolenic (C18:3) acid-did not change in both storage regimes. The lycopene and β-carotene contents increased slightly during storage (20 and 7°C), as the carotenoid-derived volatile 6-methyl-5-hepten-2-one did. The fructose and glucose concentrations did not vary significantly, while the content of total soluble solids increased during both storage regimes. No significant difference could be found between the fruits stored at 20 or 7°C for 4 days after the post-harvest handling in all the parameters analyzed, including the sensory analysis, considering all cultivars/breeding lines. A storage temperature of 7°C is not detrimental for the flavor of ripe fruits under the experimental conditions used. The genetic background of the studied cultivars/breeding lines have a higher impact on the flavor variation than the two common household storage conditions when storing ripe fruits and taking the entire post-harvest handling into account.

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein-Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail.

Successful defence of plants against colonisation by fungal pathogens depends on the ability to prevent initial penetration of the plant cell wall. Here we report that the pathogen-induced (1,3)-β-glucan cell wall polymer callose, which is deposited at sites of attempted penetration, directly interacts with the most prominent cell wall polymer, the (1,4)-β-glucan cellulose, to form a three-dimensional network at sites of attempted fungal penetration. Localisation microscopy, a super-resolution microscopy technique based on the precise localisation of single fluorescent molecules, facilitated discrimination between single polymer fibrils in this network. Overexpression of the pathogen-induced callose synthase PMR4 in the model plant Arabidopsis thaliana not only enlarged focal callose deposition and polymer network formation but also resulted in the exposition of a callose layer on the surface of the pre-existing cellulosic cell wall facing the invading pathogen. The importance of this previously unknown polymeric defence network is to prevent cell wall hydrolysis and penetration by the fungus. We anticipate our study to promote nanoscale analysis of plant-microbe interactions with a special focus on polymer rearrangements in and at the cell wall. Moreover, the general applicability of localisation microscopy in visualising polymers beyond plant research will help elucidate their biological function in complex networks.

As stated by the prevailing amyloid cascade hypothesis, Alzheimer's disease (AD) is caused by the aggregation and cerebral deposition of long amyloid-β peptide (Aβ) species, which are released from a C-terminal amyloid precursor protein fragment by γ-secretase. Mutations in its catalytic subunit presenilin-1 (PS1) increase the Aβ42 to Aβ40 ratio and are the major cause of familial AD (FAD). An opposing hypothesis states that loss of essential presenilin functions underlies the disease. A major argument for this hypothesis is the observation that the nearly inactive PS1 L435F mutant, paradoxically, causes FAD We now show that the very little Aβ generated by PS1 L435F consists primarily of Aβ43, a highly amyloidogenic species which was overlooked in previous studies of this mutant. We further demonstrate that the generation of Aβ43 is not due to a trans-dominant effect of this mutant on WT presenilin. Furthermore, we found Aβ43-containing plaques in brains of patients with this mutation. The aberrant generation of Aβ43 by this particular mutant provides a direct objection against the presenilin hypothesis.

Autosomal-dominant mutations within the gene FUS (fused in sarcoma) are responsible for 5% of familial cases of amyotrophic lateral sclerosis (ALS). The FUS protein is physiologically mainly located in the nucleus, while cytoplasmic FUS aggregates are pathological hallmarks of FUS-ALS. Data from non-neuronal cell models and/or models using heterologous expression of FUS mutants suggest cytoplasmic FUS translocation as a pivotal initial event which leads to neurodegeneration depending on a second hit. Here we present the first human model of FUS-ALS using patient-derived neurons carrying endogenous FUS mutations leading to a benign (R521C) or a more severe clinical phenotype (frameshift mutation R495QfsX527). We thereby showed that the severity of the underlying FUS mutation determines the amount of cytoplasmic FUS accumulation and cellular vulnerability to exogenous stress. Cytoplasmic FUS inclusions formed spontaneously depending on both, severity of FUS mutation and neuronal aging. These aggregates showed typical characteristics of FUS-ALS including methylated FUS. Finally, neurodegeneration was not specific to layer V cortical neurons perfectly in line with the current model of disease spreading in ALS. Our study highlights the value and usefulness of patient-derived cell models in FUS-ALS.

This study depicts relations between magnesium (Mg) transport and re-translocation, photoassimilate partitioning, cation and ion concentrations, and finally root growth of potato under different Mg supplies. Potato plants were grown in a hydroponic culture system under different Mg regimes while investigating Mg concentrations, the expression of various Mg transporters, soluble sugars, and cations and anions in source and sink organs at different growth stages. Reports from literature about the impact of Mg deficiency on root growth are inconsistent. As Mg is known to be a phloem mobile nutrient, it is expected to be re-translocated under restricted availability of Mg from source to sink organs. Thus, we assume that plants can tolerate a slight Mg restriction without severe root growth reduction. However, under severe Mg deficiency, the process of Mg re-translocation is hampered, resulting in an impaired photoassimilate partitioning, and finally root growth. This might also explain the findings of studies claiming that Mg deficiency does not impair root growth as plants of these studies likely only suffered a slight Mg restriction. Finally, this study gives indications that an interruption of the process of Mg-re-translocation in early plant growth could be an indicator for growth reductions of the plant at a later growth stage.