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.

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 \textgreater97%, 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.

The evolutionary conserved NEAT1-MALAT1 gene cluster generates large noncoding transcripts remaining nuclear, while tRNA-like transcripts (mascRNA, menRNA) enzymatically generated from these precursors translocate to the cytosol. Whereas functions have been assigned to the nuclear transcripts, data on biological functions of the small cytosolic transcripts are sparse. We previously found NEAT1-/- and MALAT1-/- mice to display massive atherosclerosis and vascular inflammation. Here, employing selective targeted disruption of menRNA or mascRNA, we investigate the tRNA-like molecules as critical components of innate immunity. CRISPR-generated human ΔmascRNA and ΔmenRNA monocytes/macrophages display defective innate immune sensing, loss of cytokine control, imbalance of growth/angiogenic factor expression impacting upon angiogenesis, and altered cell-cell interaction systems. Antiviral response, foam cell formation/oxLDL uptake, and M1/M2 polarization are defective in ΔmascRNA/ΔmenRNA macrophages, defining first biological functions of menRNA and describing new functions of mascRNA. menRNA and mascRNA represent novel components of innate immunity arising from the noncoding genome. They appear as prototypes of a new class of noncoding RNAs distinct from others (miRNAs, siRNAs) by biosynthetic pathway and intracellular kinetics. Their NEAT1-MALAT1 region of origin appears as archetype of a functionally highly integrated RNA processing system.

Aging of the skin is accompanied by cellular as well as tissue environmental changes, ultimately reducing the ability of the tissue to regenerate and adequately respond to external stressors. Macrophages are important gatekeepers of tissue homeostasis, and it has been reported that their number and phenotype change during aging in a site-specific manner. How aging affects human skin macrophages and what implications this has for the aging process in the tissue are still not fully understood. Using single-cell RNA-sequencing analysis, we show that there is at least a 50% increase of macrophages in human aged skin, which appear to have developed from monocytes and exhibit more proinflammatory M1-like characteristics. In contrast, the cell-intrinsic ability of aged monocytes to differentiate into M1 macrophages was reduced. Using coculture experiments with aged dermal fibroblasts, we show that it is the aged microenvironment that drives a more proinflammatory phenotype of macrophages in the skin. This proinflammatory M1-like phenotype in turn negatively influenced the expression of extracellular matrix proteins by fibroblasts, emphasizing the impact of the aged macrophages on the skin phenotype.

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.

Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

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.

[This corrects the article DOI: 10.3389/fgene.2022.931017.].

Tafazzin-an acyltransferase-is involved in cardiolipin (CL) remodeling. CL is associated with mitochondrial function, structure and more recently with cell proliferation. Various tafazzin isoforms exist in humans. The role of these isoforms in cardiolipin remodeling is unknown. Aim of this study was to investigate if specific isoforms like Δ5 can restore the wild type phenotype with respect to CL composition, cellular proliferation and gene expression profile. In addition, we aimed to determine the molecular mechanism by which tafazzin can modulate gene expression by applying promoter analysis and (Ingenuity Pathway Analyis) IPA to genes regulated by TAZ-deficiency. Expression of Δ5 and rat full length TAZ in C6-TAZ- cells could fully restore CL composition and-as proven for Δ5-this is naturally associated with restoration of mitochondrial respiration. A similar restoration of CL-composition could not be observed after re-expression of an enzymatically dead full-length rat TAZ (H69L; TAZMut). Re-expression of only rat full length TAZ could restore proliferation rate. Surprisingly, the Δ5 variant failed to restore wild-type proliferation. Further, as expected, re-expression of the TAZMut variant completely failed to reverse the gene expression changes, whereas re-expression of the TAZ-FL variant largely did so and the Δ5 variant to somewhat less extent. Very likely TAZ-deficiency provokes substantial long-lasting changes in cellular lipid metabolism which contribute to changes in proliferation and gene expression, and are not or only very slowly reversible.

Alternative splicing (AS) is a major mechanism for gene expression in eukaryotes, increasing proteome diversity but also regulating transcriptome abundance. High temperatures have a strong impact on the splicing profile of many genes and therefore AS is considered as an integral part of heat stress response. While many studies have established a detailed description of the diversity of the RNAome under heat stress in different plant species and stress regimes, little is known on the underlying mechanisms that control this temperature-sensitive process. AS is mainly regulated by the activity of splicing regulators. Changes in the abundance of these proteins through transcription and AS, post-translational modifications and interactions with exonic and intronic cis-elements and core elements of the spliceosomes modulate the outcome of pre-mRNA splicing. As a major part of pre-mRNAs are spliced co-transcriptionally, the chromatin environment along with the RNA polymerase II elongation play a major role in the regulation of pre-mRNA splicing under heat stress conditions. Despite its importance, our understanding on the regulation of heat stress sensitive AS in plants is scarce. In this review, we summarize the current status of knowledge on the regulation of AS in plants under heat stress conditions. We discuss possible implications of different pathways based on results from non-plant systems to provide a perspective for researchers who aim to elucidate the molecular basis of AS under high temperatures.

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 β-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic β-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope β-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 β-strands. This process requires the action of translocon of the outer chloroplast (TOC) membrane. After translocation into the intermembrane space, β-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic β-barrel proteins is affected by mutation of the last β-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic β-barrel protein import.

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.

Proper and size selective blood filtration in the kidney depends on an intact morphology of podocyte foot processes. Effacement of interdigitating podocyte foot processes in the glomeruli causes a leaky filtration barrier resulting in proteinuria followed by the development of chronic kidney diseases. Since the function of the filtration barrier is depending on a proper actin cytoskeleton, we studied the role of the important actin-binding protein palladin for podocyte morphology. Podocyte-specific palladin knockout mice on a C57BL/6 genetic background (PodoPalldBL/6-/-) were back crossed to a 129 genetic background (PodoPalld129-/-) which is known to be more sensitive to kidney damage. Then we analyzed the morphological changes of glomeruli and podocytes as well as the expression of the palladin-binding partners Pdlim2, Lasp-1, Amotl1, ezrin and VASP in 6 and 12 months old mice. PodoPalld129-/- mice in 6 and 12 months showed a marked dilatation of the glomerular tuft and a reduced expression of the mesangial marker protein integrin α8 compared to controls of the same age. Furthermore, ultrastructural analysis showed significantly more podocytes with morphological deviations like an enlarged sub-podocyte space and regions with close contact to parietal epithelial cells. Moreover, PodoPalld129-/- of both age showed a severe effacement of podocyte foot processes, a significantly reduced expression of pLasp-1 and Pdlim2, and significantly reduced mRNA expression of Pdlim2 and VASP, three palladin-interacting proteins. Taken together, the results show that palladin is essential for proper podocyte morphology in mice with a 129 background.

Multi-drug resistant (MDR), gram-negative Enterobacteriaceae, such as Escherichia coli (E. coli) limit therapeutic options and increase morbidity, mortality, and treatment costs worldwide. They pose a serious burden on healthcare systems, especially in developing countries like Rwanda. Several studies have shown the effects caused by the global spread of extended-spectrum beta-lactamase (ESBL)-producing E. coli. However, limited data is available on transmission dynamics of these pathogens and the mobile elements they carry in the context of clinical and community locations in Sub-Saharan Africa. Here, we examined 120 ESBL-producing E. coli strains from patients hospitalized in the University Teaching Hospital of Butare (Rwanda), their attending caregivers as well as associated community members and livestock. Based on whole-genome analysis, the genetic diversification and phylogenetics were assessed. Moreover, the content of carried plasmids was characterized and investigated for putative transmission among strains, and for their potential role as drivers for the spread of antibiotic resistance. We show that among the 30 different sequence types (ST) detected were the pandemic clonal lineages ST131, ST648 and ST410, which combine high-level antimicrobial resistance with virulence. In addition to the frequently found resistance genes bla CTX-M-15 , tet(34), and aph(6)-Id, we identified csg genes, which are required for curli fiber synthesis and thus biofilm formation. Numerous strains harbored multiple virulence-associated genes (VAGs) including pap (P fimbriae adhesion cluster), fim (type I fimbriae) and chu (Chu heme uptake system). Furthermore, we found phylogenetic relationships among strains from patients and their caregivers or related community members and animals, which indicates transmission of pathogens. Also, we demonstrated the presence and potential transfer of identical/similar ESBL-plasmids in different strains from the Rwandan setting and when compared to an external plasmid. This study highlights the circulation of clinically relevant, pathogenic ESBL-producing E. coli among patients, caregivers and the community in Rwanda. Combining antimicrobial resistance with virulence in addition to the putative exchange of mobile genetic elements among bacterial pathogens poses a significant risk around the world.

Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.

Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification.

Cellular transitions during development and stress response depend on coordinated transcriptomic and proteomic alterations. Pollen is particular because its development is a complex process that includes meiotic and mitotic divisions which causes a high heat sensitivity of these cells. Development and stress response are accompanied by a reprogramming of the transcriptome, e.g. by post-transcriptional regulation via miRNAs. We identified known and potentially novel miRNAs in the transcriptome of developing and heat-stressed pollen of Solanum lycopersicum (tomato). The prediction of target mRNAs yielded an equal number of predicted target-sites in CDS and 3’UTR regions of target mRNAs. The result enabled the postulation of a possible link between miRNAs and a fine-tuning of transcription factor abundance during pollen development. miRNAs seem to play a role in the pollen heat stress response as well. We identified several heat stress transcription factors and heat shock proteins as putative targets of miRNAs in response to heat stress, thereby placing these miRNAs as important elements of thermotolerance. Moreover, for members of the AP2, SBP and ARF family members we could predict a miRNA-mediated regulation during development via the miR172, mir156 and mir160-family strengthening the current concept of a cross-connection between development and stress response in plants.

Transcriptional reprograming after the exposure of plants to elevated temperatures is a hallmark of stress response which is required for the manifestation of thermotolerance. Central transcription factors regulate the stress survival and recovery mechanisms and many of the core responses controlled by these factors are well described. In turn, pathways and specific genes contributing to variations in the thermotolerance capacity even among closely related plant genotypes are not well defined. A seedling-based assay was developed to directly compare the growth and transcriptome response to heat stress in four tomato genotypes with contrasting thermotolerance. The conserved and the genotype-specific alterations of mRNA abundance in response to heat stress were monitored after exposure to three different temperatures. The transcripts of the majority of genes behave similarly in all genotypes, including the majority of heat stress transcription factors and heat shock proteins, but also genes involved in photosynthesis and mitochondrial ATP production. In turn, genes involved in hormone and RNA-based regulation, such as auxin- and ethylene-related genes, or transcription factors like HsfA6b, show a differential regulation that associates with the thermotolerance pattern. Our results provide an inventory of genes likely involved in core and genotype-dependent heat stress response mechanisms with putative role in thermotolerance in tomato seedlings.

The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of β-barrel-type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of β-barrel-type proteins. At present, only the processing of Toc75-III, the β-barrel-type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75-V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75-V/OEP80 is processed as well, we reinvestigated the processing in a protoplast-based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N-terminal domain. Thus, Toc75-V contains a cleavable N-terminal signal and exposes its polypeptide transport-associated domains to the intermembrane space of plastids, where it likely interacts with its substrates.