Investigating FSGS-like Injury in Zebrafish Larvae by Nifurpirinol: Efficacy and Molecular Insight.

Identifying effective drugs for focal segmental glomerulosclerosis (FSGS) treatment holds significant importance. Our high-content drug screening on zebrafish larvae relies on nitroreductase/metronidazole (NTR/MTZ)-induced podocyte ablation to generate FSGS-like injury. A crucial factor for successful drug screenings is minimizing variability in injury induction. For this, we introduce Nifurpirinol (NFP) as a more reliable prodrug for targeted podocyte depletion. NFP showed a 2.3-fold increase in efficiency at concentrations 1600-fold lower compared to MTZ-mediated injury induction. Integration into the screening workflow validated its suitability for the high-content drug screening. The presence of crucial FSGS hallmarks such as podocyte foot process effacement, proteinuria, and activation of parietal epithelial cells, were found. After the isolation of the glomeruli from the larvae, we identified essential pathways by proteomic analysis. This study shows that NFP serves as a highly effective prodrug to induce the FSGS-like disease in zebrafish larvae and is well-suited for a high-content drug screening to identify new candidates for the treatment of FSGS.

Insertion of plastidic ß-barrel proteins into the outer envelopes of plastids involves an intermembrane space intermediate formed with Toc75-V/OEP80.

The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic ß-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic ß-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope ß-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for the translocation of pea proteins across the outer envelope membrane is present within the six N-terminal ß-strands. This process requires the action of translocon of the outer chloroplast (TOC) membrane. After translocation into the intermembrane space, ß-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic ß-barrel proteins is affected by mutation of the last ß-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic ß-barrel protein import.

Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis.

Arabidopsis contains hundreds of ribosomal DNA copies organized within the nucleolar organizing regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1-4, based on the polymorphisms of 3' external transcribed sequences. The variants are known to be differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~ 25 nt from predominantly NOR4 ribosomal DNA copies, obtaining mosaic mutational events on ~ 5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies by elevated expression of VAR1 in mutants and further associated single-nucleotide variants, thus, resulting in altered rRNA sub-population. Furthermore, the mutants exhibited rRNA maturation defects specifically in the minor pathway typified by 32S pre-rRNA accumulation. Density-gradient fractionation and subsequent RT-PCR of rRNA analyses revealed that mutated copies were not incorporated into the translating ribosomes. The mutants in addition displayed an elevated autophagic flux as shown by the autophagic marker GFP-ATG8e, likely related to ribophagy.

A Novel High-Content Screening Assay Identified Belinostat as Protective in a FSGS-Like Zebrafish Model.

BACKGROUND: FSGS affects the complex three-dimensional morphology of podocytes, resulting in loss of filtration barrier function and the development of sclerotic lesions. Therapies to treat FSGS are limited, and podocyte-specific drugs are unavailable. To address the need for treatments to delay or stop FSGS progression, researchers are exploring the repurposing of drugs that have been approved by the US Food and Drug Administration (FDA) for other purposes. METHODS: To identify drugs with potential to treat FSGS, we used a specific zebrafish screening strain to combine a high-content screening (HCS) approach with an in vivo model. This zebrafish screening strain expresses nitroreductase and the red fluorescent protein mCherry exclusively in podocytes (providing an indicator for podocyte depletion), as well as a circulating 78 kDa vitamin D-binding enhanced green fluorescent protein fusion protein (as a readout for proteinuria). To produce FSGS-like lesions in the zebrafish, we added 80 µ M metronidazole into the fish water. We used a specific screening microscope in conjunction with advanced image analysis methods to screen a library of 138 drugs and compounds (including some FDA-approved drugs) for podocyte-protective effects. Promising candidates were validated to be suitable for translational studies. RESULTS: After establishing this novel in vivo HCS assay, we identified seven drugs or compounds that were protective in our FSGS-like model. Validation experiments confirmed that the FDA-approved drug belinostat was protective against larval FSGS. Similar pan-histone deacetylase inhibitors also showed potential to reproduce this effect. CONCLUSIONS: Using an FSGS-like zebrafish model, we developed a novel in vivo HCS assay that identified belinostat and related pan-histone deacetylase inhibitors as potential candidates for treating FSGS.

MncR: Late Integration Machine Learning Model for Classification of ncRNA Classes Using Sequence and Structural Encoding.

Non-coding RNA (ncRNA) classes take over important housekeeping and regulatory functions and are quite heterogeneous in terms of length, sequence conservation and secondary structure. High-throughput sequencing reveals that the expressed novel ncRNAs and their classification are important to understand cell regulation and identify potential diagnostic and therapeutic biomarkers. To improve the classification of ncRNAs, we investigated different approaches of utilizing primary sequences and secondary structures as well as the late integration of both using machine learning models, including different neural network architectures. As input, we used the newest version of RNAcentral, focusing on six ncRNA classes, including lncRNA, rRNA, tRNA, miRNA, snRNA and snoRNA. The late integration of graph-encoded structural features and primary sequences in our MncR classifier achieved an overall accuracy of >97%, which could not be increased by more fine-grained subclassification. In comparison to the actual best-performing tool ncRDense, we had a minimal increase of 0.5% in all four overlapping ncRNA classes on a similar test set of sequences. In summary, MncR is not only more accurate than current ncRNA prediction tools but also allows the prediction of long ncRNA classes (lncRNAs, certain rRNAs) up to 12.000 nts and is trained on a more diverse ncRNA dataset retrieved from RNAcentral.

Machine Learning Based Methods and Best Practices of microRNA-Target Prediction and Validation.

Within the last years, more and more noncoding RNAs (ncRNAs) became the focal point to understand cell regulatory mechanisms because one class of ncRNAs, microRNAs (miRNAs), plays an essential role in translation repression or degradation of specific mRNAs and is implicated in disease etiology. miRNAs can serve as oncomiRs (oncogenic miRNAs) and tumor suppressor miRNAs, thus, miRNA therapeutics in clinical trials have become a vital component with respect to cancer treatment. To circumvent side-effects and allow an accurate effect it is crucial to accurately predict miRNAs and their mRNA targets. Over the last two decades, different approaches for miRNA prediction as well as miRNA target prediction have been developed and improved based on sequence and structure features. Nowadays, the abundance of high-throughput sequencing data and databases of miRNAs and miRNA targets from different species allow the training, testing, and validation of predicted miRNAs and miRNA targets with machine learning methods. This book chapter focuses on the important requirements for miRNA target prediction tools using ML like common features used for miRNA-binding site prediction. Furthermore, best practices for the prediction and validation of miRNA-mRNA targets are presented and set in the context of possible applications for cancer diagnosis and therapeutics.

Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

Plant-specific ribosome biogenesis factors in Arabidopsis thaliana with essential function in rRNA processing.

rRNA processing and assembly of ribosomal proteins during maturation of ribosomes involve many ribosome biogenesis factors (RBFs). Recent studies identified differences in the set of RBFs in humans and yeast, and the existence of plant-specific RBFs has been proposed as well. To identify such plant-specific RBFs, we characterized T-DNA insertion mutants of 15 Arabidopsis thaliana genes encoding nuclear proteins with nucleotide binding properties that are not orthologues to yeast or human RBFs. Mutants of nine genes show an altered rRNA processing ranging from inhibition of initial 35S pre-rRNA cleavage to final maturation events like the 6S pre-rRNA processing. These phenotypes led to their annotation as 'involved in rRNA processing' - IRP. The irp mutants are either lethal or show developmental and stress related phenotypes. We identified IRPs for maturation of the plant-specific precursor 5'-5.8S and one affecting the pathway with ITS2 first cleavage of the 35S pre-rRNA transcript. Moreover, we realized that 5'-5.8S processing is essential, while a mutant causing 6S accumulation shows only a weak phenotype. Thus, we demonstrate the importance of the maturation of the plant-specific precursor 5'-5.8S for plant development as well as the occurrence of an ITS2 first cleavage pathway in fast dividing tissues.

Effect of thermospermine on expression profiling of different gene using massive analysis of cDNA ends (MACE) and vascular maintenance in Arabidopsis.

Arabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) was significantly repressed by low dose(s) of T-Spm but not by other polyamines. To figure out the underlying mechanism, massive analysis of 3'-cDNA ends was performed. Low dose of T-Spm treatment modulates more than two fold expression 1,398 genes in WT compared to 3186 genes in Atpao5-2. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose T-Spm-treated Atpao5-2, in comparison to other pathways such as TCA cycle-, amino acid- metabolisms and photosynthesis. The cell wall pectin metabolism, cell wall proteins and degradation process were highly modulated. Intriguingly Fe-deficiency responsive genes and drought stress-induced genes were also up-regulated, suggesting the importance of thermospermi'ne flux on regulation of gene network. Histological observation showed that the vascular system of the joint part between stem and leaves was structurally dissociated, indicating its involvement in vascular maintenance. Endogenous increase in T-Spm and reduction in H2O2 contents were found in mutant grown in T-Spm containing media. The results indicate that T-Spm homeostasis by a fine tuned balance of its synthesis and catabolism is important for maintaining gene regulation network and the vascular system in plants.

The repressor and co-activator HsfB1 regulates the major heat stress transcription factors in tomato.

Plants code for a multitude of heat stress transcription factors (Hsfs). Three of them act as central regulators of heat stress (HS) response in tomato (Solanum lycopersicum). HsfA1a regulates the initial response, and HsfA2 controls acquired thermotolerance. HsfB1 is a transcriptional repressor but can also act as co-activator of HsfA1a. Currently, the mode of action and the relevance of the dual function of HsfB1 remain elusive. We examined this in HsfB1 overexpression or suppression transgenic tomato lines. Proteome analysis revealed that HsfB1 overexpression stimulates the co-activator function of HsfB1 and consequently the accumulation of HS-related proteins under non-stress conditions. Plants with enhanced levels of HsfB1 show aberrant growth and development but enhanced thermotolerance. HsfB1 suppression has no significant effect prior to stress. Upon HS, HsfB1 suppression strongly enhances the induction of heat shock proteins due to the higher activity of other HS-induced Hsfs, resulting in increased thermotolerance compared with wild-type. Thereby, HsfB1 acts as co-activator of HsfA1a for several Hsps, but as a transcriptional repressor on other Hsfs, including HsfA1b and HsfA2. The dual function explains the activation of chaperones to enhance protection and regulate the balance between growth and stress response upon deviations from the homeostatic levels of HsfB1.

Co-orthologues of ribosome biogenesis factors in A. thaliana are differentially regulated by transcription factors.

KEY MESSAGE: Different genes coding for one ribosome biogenesis factor are differentially expressed and are likely under the control of distinct transcription factors, which contributes to the regulatory space for ribosome maturation. Maturation of ribosomes including rRNA processing and modification, rRNA folding and ribosome protein association requires the function of many ribosome biogenesis factors (RBFs). Recent studies document plant-specific variations of the generally conserved process of ribosome biogenesis. For instance, distinct rRNA maturation pathways and intermediates have been identified, the existence of plant specific RBFs has been proposed and several RBFs are encoded by multiple genes. The latter in combination with the discussed ribosome heterogeneity points to a possible function of the different proteins representing one RBF in diversification of ribosomal compositions. Such factor-based regulation would require a differential regulation of their expression, may be even controlled by different transcription factors. We analyzed the expression profiles of genes coding for putative RBFs and transcription factors. Most of the genes coding for RBFs are expressed in a comparable manner, while different genes coding for a single RBF are often differentially expressed. Based on a selected set of genes we document a function of the transcription factors AtMYC1, AtMYC2, AtbHLH105 and AtMYB26 on the regulation of different RBFs. Moreover, on the example of the RBFs LSG1 and BRX1, both encoded by two genes, we give a first hint on a differential transcription factor dependence of expression. Consistent with this observation, the phenotypic analysis of RBF mutants suggests a relation between LSG1-1 and BRX1-1 expression and the transcription factor MYC1. In summary, we propose that the multiple genes coding for one RBF are required to enlarge the regulatory space for ribosome biogenesis.

The coupling of transcriptome and proteome adaptation during development and heat stress response of tomato pollen.

BACKGROUND: Pollen development is central for plant reproduction and is assisted by changes of the transcriptome and proteome. At the same time, pollen development and viability is largely sensitive to stress, particularly to elevated temperatures. The transcriptomic and proteomic changes during pollen development and of different stages in response to elevated temperature was targeted to define the underlying molecular principles. RESULTS: The analysis of the transcriptome and proteome of Solanum lycopersicum pollen at tetrad, post-meiotic and mature stage before and after heat stress yielded a decline of the transcriptome but an increase of the proteome size throughout pollen development. Comparison of the transcriptome and proteome led to the discovery of two modes defined as direct and delayed translation. Here, genes of distinct functional processes are under the control of direct and delayed translation. The response of pollen to elevated temperature occurs rather at proteome, but not as drastic at the transcriptome level. Heat shock proteins, proteasome subunits, ribosomal proteins and eukaryotic initiation factors are most affected. On the example of heat shock proteins we demonstrate a decoupling of transcript and protein levels as well as a distinct regulation between the developmental stages. CONCLUSIONS: The transcriptome and proteome of developing pollen undergo drastic changes in composition and quantity. Changes at the proteome level are a result of two modes assigned as direct and delayed translation. The response of pollen to elevated temperature is mainly regulated at the proteome level, whereby proteins related to synthesis and degradation of proteins are most responsive and might play a central role in the heat stress response of pollen.

HsfA2 Controls the Activity of Developmentally and Stress-Regulated Heat Stress Protection Mechanisms in Tomato Male Reproductive Tissues.

Male reproductive tissues are more sensitive to heat stress (HS) compared to vegetative tissues, but the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection from HS In tomato (Solanum lycopersicum), HsfA2 acts as coactivator of HsfA1a and is one of the major Hsfs accumulating in response to elevated temperatures. The contribution of HsfA2 in heat stress response (HSR) and thermotolerance was investigated in different tissues of transgenic tomato plants with suppressed HsfA2 levels (A2AS). Global transcriptome analysis and immunodetection of two major Hsps in vegetative and reproductive tissues showed that HsfA2 regulates subsets of HS-induced genes in a tissue-specific manner. Accumulation of HsfA2 by a moderate HS treatment enhances the capacity of seedlings to cope with a subsequent severe HS, suggesting an important role for HsfA2 in regulating acquired thermotolerance. In pollen, HsfA2 is an important coactivator of HsfA1a during HSR HsfA2 suppression reduces the viability and germination rate of pollen that received the stress during the stages of meiosis and microspore formation but had no effect on more advanced stages. In general, pollen meiocytes and microspores are characterized by increased susceptibility to HS due to their lower capacity to induce a strong HSR This sensitivity is partially mitigated by the developmentally regulated expression of HsfA2 and several HS-responsive genes mediated by HsfA1a under nonstress conditions. Thereby, HsfA2 is an important factor for the priming process that sustains pollen thermotolerance during microsporogenesis.

The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21.

Translation fidelity and efficiency require multiple ribosomal (r)RNA modifications that are mostly mediated by small nucleolar (sno)RNPs during ribosome production. Overlapping basepairing of snoRNAs with pre-rRNAs often necessitates sequential and efficient association and dissociation of the snoRNPs, however, how such hierarchy is established has remained unknown so far. Here, we identify several late-acting snoRNAs that bind pre-40S particles in human cells and show that their association and function in pre-40S complexes is regulated by the RNA helicase DDX21. We map DDX21 crosslinking sites on pre-rRNAs and show their overlap with the basepairing sites of the affected snoRNAs. While DDX21 activity is required for recruitment of the late-acting snoRNAs SNORD56 and SNORD68, earlier snoRNAs are not affected by DDX21 depletion. Together, these observations provide an understanding of the timing and ordered hierarchy of snoRNP action in pre-40S maturation and reveal a novel mode of regulation of snoRNP function by an RNA helicase in human cells.

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase.

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the "foot" region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.

Proteome distribution between nucleoplasm and nucleolus and its relation to ribosome biogenesis in Arabidopsis thaliana.

Ribosome biogenesis is an essential process initiated in the nucleolus. In eukaryotes, multiple ribosome biogenesis factors (RBFs) can be found in the nucleolus, the nucleus and in the cytoplasm. They act in processing, folding and modification of the pre-ribosomal (r)RNAs, incorporation of ribosomal proteins (RPs), export of pre-ribosomal particles to the cytoplasm, and quality control mechanisms. Ribosome biogenesis is best established for Saccharomyces cerevisiae. Plant ortholog assignment to yeast RBFs revealed the absence of about 30% of the yeast RBFs in plants. In turn, few plant specific proteins have been identified by biochemical experiments to act in plant ribosome biogenesis. Nevertheless, a complete inventory of plant RBFs has not been established yet. We analyzed the proteome of the nucleus and nucleolus of Arabidopsis thaliana and the post-translational modifications of these proteins. We identified 1602 proteins in the nucleolar and 2544 proteins in the nuclear fraction with an overlap of 1429 proteins. For a randomly selected set of proteins identified by the proteomic approach we confirmed the localization inferred from the proteomics data by the localization of GFP fusion proteins. We assigned the identified proteins to various complexes and functions and found about 519 plant proteins that have a potential to act as a RBFs, but which have not been experimentally characterized yet. Last, we compared the distribution of RBFs and RPs in the various fractions with the distribution established for yeast.

50 years of amino acid hydrophobicity scales: revisiting the capacity for peptide classification.

BACKGROUND: Physicochemical properties are frequently analyzed to characterize protein-sequences of known and unknown function. Especially the hydrophobicity of amino acids is often used for structural prediction or for the detection of membrane associated or embedded ß-sheets and α-helices. For this purpose many scales classifying amino acids according to their physicochemical properties have been defined over the past decades. In parallel, several hydrophobicity parameters have been defined for calculation of peptide properties. We analyzed the performance of separating sequence pools using 98 hydrophobicity scales and five different hydrophobicity parameters, namely the overall hydrophobicity, the hydrophobic moment for detection of the α-helical and ß-sheet membrane segments, the alternating hydrophobicity and the exact ß-strand score. RESULTS: Most of the scales are capable of discriminating between transmembrane α-helices and transmembrane ß-sheets, but assignment of peptides to pools of soluble peptides of different secondary structures is not achieved at the same quality. The separation capacity as measure of the discrimination between different structural elements is best by using the five different hydrophobicity parameters, but addition of the alternating hydrophobicity does not provide a large benefit. An in silico evolutionary approach shows that scales have limitation in separation capacity with a maximal threshold of 0.6 in general. We observed that scales derived from the evolutionary approach performed best in separating the different peptide pools when values for arginine and tyrosine were largely distinct from the value of glutamate. Finally, the separation of secondary structure pools via hydrophobicity can be supported by specific detectable patterns of four amino acids. CONCLUSION: It could be assumed that the quality of separation capacity of a certain scale depends on the spacing of the hydrophobicity value of certain amino acids. Irrespective of the wealth of hydrophobicity scales a scale separating all different kinds of secondary structures or between soluble and transmembrane peptides does not exist reflecting that properties other than hydrophobicity affect secondary structure formation as well. Nevertheless, application of hydrophobicity scales allows distinguishing between peptides with transmembrane α-helices and ß-sheets. Furthermore, the overall separation capacity score of 0.6 using different hydrophobicity parameters could be assisted by pattern search on the protein sequence level for specific peptides with a length of four amino acids.

The evolution of the ribosome biogenesis pathway from a yeast perspective.

Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and insights are even more limited for archaea. Starting from 255 yeast RBFs, we integrated ortholog searches, domain architecture comparisons and, in part, manual curation to investigate the inventories of RBF candidates in 261 eukaryotes, 26 archaea and 57 bacteria. The resulting phylogenetic profiles reveal the evolutionary ancestry of the yeast pathway. The oldest core comprising 20 RBF lineages dates back to the last universal common ancestor, while the youngest 20 factors are confined to the Saccharomycotina. On this basis, we outline similarities and differences of ribosome biogenesis across contemporary species. Archaea, so far a rather uncharted domain, possess 38 well-supported RBF candidates of which some are known to form functional sub-complexes in yeast. This provides initial evidence that ribosome biogenesis in eukaryotes and archaea follows similar principles. Within eukaryotes, RBF repertoires vary considerably. A comparison of yeast and human reveals that lineage-specific adaptation via RBF exclusion and addition characterizes the evolution of this ancient pathway.

Chaperone network composition in Solanum lycopersicum explored by transcriptome profiling and microarray meta-analysis.

Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS transcription factors (Hsfs). The role of Hsfs and Hsps in HS response in tomato was initially examined by transcriptome analysis using the massive analysis of cDNA ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points towards two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato.

Agent-based modeling to estimate the impact of lockdown scenarios and events on a pandemic exemplified on SARS-CoV-2.

In actual pandemic situations like COVID-19, it is important to understand the influence of single mitigation measures as well as combinations to create most dynamic impact for lockdown scenarios. Therefore we created an agent-based model (ABM) to simulate the spread of SARS-CoV-2 in an abstract city model with several types of places and agents. In comparison to infection numbers in Germany our ABM could be shown to behave similarly during the first wave. In our model, we implemented the possibility to test the effectiveness of mitigation measures and lockdown scenarios on the course of the pandemic. In this context, we focused on parameters of local events as possible mitigation measures and ran simulations, including varying size, duration, frequency and the proportion of events. The majority of changes to single event parameters, with the exception of frequency, showed only a small influence on the overall course of the pandemic. By applying different lockdown scenarios in our simulations, we could observe drastic changes in the number of infections per day. Depending on the lockdown strategy, we even observed a delayed peak in infection numbers of the second wave. As an advantage of the developed ABM, it is possible to analyze the individual risk of single agents during the pandemic. In contrast to standard or adjusted ODEs, we observed a 21% (with masks) / 48% (without masks) increased risk for single reappearing participants on local events, with a linearly increasing risk based on the length of the events.