Glutamyl-prolyl-tRNA synthetase (EPRS1) drives tubulointerstitial nephritis-induced fibrosis by enhancing T cell proliferation and activity.

Toxin- and drug-induced tubulointerstitial nephritis (TIN), characterized by interstitial infiltration of immune cells, frequently necessitates dialysis for patients due to irreversible fibrosis. However, agents modulating interstitial immune cells are lacking. Here, we addressed whether the housekeeping enzyme glutamyl-prolyl-transfer RNA synthetase 1 (EPRS1), responsible for attaching glutamic acid and proline to transfer RNA, modulates immune cell activity during TIN and whether its pharmacological inhibition abrogates fibrotic transformation. The immunological feature following TIN induction by means of an adenine-mixed diet was infiltration of EPRS1high T cells, particularly proliferating T and γδ T cells. The proliferation capacity of both CD4+ and CD8+ T cells, along with interleukin-17 production of γδ T cells, was higher in the kidneys of TIN-induced Eprs1+/+ mice than in the kidneys of TIN-induced Eprs1+/- mice. This discrepancy contributed to the fibrotic amelioration observed in kidneys of Eprs1+/- mice. TIN-induced fibrosis was also reduced in Rag1-/- mice adoptively transferred with Eprs1+/- T cells compared to the Rag1-/- mice transferred with Eprs1+/+ T cells. The use of an EPRS1-targeting small molecule inhibitor (bersiporocin) under clinical trials to evaluate its therapeutic potential against idiopathic pulmonary fibrosis alleviated immunofibrotic aggravation in TIN. EPRS1 expression was also observed in human kidney tissues and blood-derived T cells, and high expression was associated with worse patient outcomes. Thus, EPRS1 may emerge as a therapeutic target in toxin- and drug-induced TIN, modulating the proliferation and activity of infiltrated T cells.

In-depth proteome analysis of brain tissue from Ewsr1 knockout mouse by multiplexed isobaric tandem mass tag labeling.

EWS RNA binding protein 1 (EWSR1) is a multifunctional protein whose epigenetic signatures contribute to the pathogenesis of various human diseases, such as neurodegenerative disorders, skin development, and tumorigenic processes. However, the specific cellular functions and physiological characteristics of EWSR1 remain unclear. In this study, we used quantitative mass spectrometry-based proteomics with tandem mass tag labeling to investigate the global proteome changes in brain tissue in Ewsr1 knockout and wild-type mice. From 9115 identified proteins, we selected 118 differentially expressed proteins, which is common to three quantitative data processing strategies including only protein level normalizations and spectrum-protein level normalization. Bioinformatics analysis of these common differentially expressed proteins revealed that proteins up-regulated in Ewsr1 knockout mouse are mostly related to the positive regulation of bone remodeling and inflammatory response. The down-regulated proteins were associated with the regulation of neurotransmitter levels or amino acid metabolic processes. Collectively, these findings provide insight into the physiological function and pathogenesis of EWSR1 on protein level. Better understanding of EWSR1 and its protein interactions will advance the field of clinical research into neuronal disorders. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD026994.

Peroxiredoxin 5 regulates osteogenic differentiation through interaction with hnRNPK during bone regeneration.

Peroxiredoxin 5 (Prdx5) is involved in pathophysiological regulation via the stress-induced cellular response. However, its function in the bone remains largely unknown. Here, we show that Prdx5 is involved in osteoclast and osteoblast differentiation, resulting in osteoporotic phenotypes in Prdx5 knockout (Prdx5Ko) male mice. To investigate the function of Prdx5 in the bone, osteoblasts were analyzed through immunoprecipitation (IP) and liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) methods, while osteoclasts were analyzed through RNA-sequencing. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as a potential binding partner of Prdx5 during osteoblast differentiation in vitro. Prdx5 acts as a negative regulator of hnRNPK-mediated osteocalcin (Bglap) expression. In addition, transcriptomic analysis revealed that in vitro differentiated osteoclasts from the bone marrow-derived macrophages of Prdx5Ko mice showed enhanced expression of several osteoclast-related genes. These findings indicate that Prdx5 might contribute to the maintenance of bone homeostasis by regulating osteoblast differentiation. This study proposes a new function of Prdx5 in bone remodeling that may be used in developing therapeutic strategies for bone diseases.

Identification of Novel Genes for Cell Fusion during Osteoclast Formation.

Osteoclasts are derived from hematopoietic stem cells. Monocyte preosteoclasts obtain resorbing activity via cell-cell fusion to generate multinucleated cells. However, the mechanisms and molecules involved in the fusion process are poorly understood. In this study, we performed RNA sequencing with single nucleated cells (SNCs) and multinucleated cells (MNCs) to identify the fusion-specific genes. The SNCs and MNCs were isolated under the same conditions during osteoclastogenesis with the receptor activator of nuclear factor-κB ligand (RANKL) administration. Based on this analysis, the expression of seven genes was found to be significantly increased in MNCs but decreased in SNCs, compared to that in bone marrow-derived macrophages (BMMs). We then generated knockout macrophage cell lines using a CRISPR-Cas9 genome-editing tool to examine their function during osteoclastogenesis. Calcrl-, Marco-, or Ube3a-deficient cells could not develop multinucleated giant osteoclasts upon RANKL stimulation. However, Tmem26-deficient cells fused more efficiently than control cells. Our findings demonstrate that Calcrl, Marco, and Ube3a are novel determinants of osteoclastogenesis, especially with respect to cell fusion, and highlight potential targets for osteoporosis therapy.

RBFOX2-regulated TEAD1 alternative splicing plays a pivotal role in Hippo-YAP signaling.

Alternative pre-mRNA splicing is key to proteome diversity; however, the biological roles of alternative splicing (AS) in signaling pathways remain elusive. Here, we focus on TEA domain transcription factor 1 (TEAD1), a YAP binding factor in the Hippo signaling pathway. Public database analyses showed that expression of YAP-TEAD target genes negatively correlated with the expression of a TEAD1 isoform lacking exon 6 (TEAD1ΔE6) but did not correlate with overall TEAD1 expression. We confirmed that the transcriptional activity and oncogenic properties of the full-length TEAD1 isoform were greater than those of TEAD1ΔE6, with the difference in transcription related to YAP interaction. Furthermore, we showed that RNA-binding Fox-1 homolog 2 (RBFOX2) promoted the inclusion of TEAD1 exon 6 via binding to the conserved GCAUG element in the downstream intron. These results suggest a regulatory mechanism of RBFOX2-mediated TEAD1 AS and provide insight into AS-specific modulation of signaling pathways.

Pulmonary fibrosis model using micro-CT analyzable human PSC-derived alveolar organoids containing alveolar macrophage-like cells.

Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68+ AMφ-like cells were spatially organized on the luminal epithelial surface of alveolar hLOs. Bleomycin-treated alveolar hLOs showed upregulated expression of fibrosis-related markers and extracellular matrix deposits in the alveolar sacs. Alveolar hLOs also showed structural alterations such as excessive tissue fraction under bleomycin treatment. Therefore, we suggest that micro-CT analyzable PSC-derived alveolar hLOs are a promising in vitro model to predict lung toxicity manifestations, including fibrosis.

Discovery of Proteins Responsible for Resistance to Three Chemotherapy Drugs in Breast Cancer Cells Using Proteomics and Bioinformatics Analysis.

Chemoresistance is a daunting obstacle to the effective treatment of breast cancer patients receiving chemotherapy. Although the mechanism of chemotherapy drug resistance has been explored broadly, the precise mechanism at the proteome level remains unclear. Especially, comparative studies between widely used anticancer drugs in breast cancer are very limited. In this study, we employed proteomics and bioinformatics approaches on chemoresistant breast cancer cell lines to understand the underlying resistance mechanisms that resulted from doxorubicin (DR), paclitaxel (PR), and tamoxifen (TAR). In total, 10,385 proteins were identified and quantified from three TMT 6-plex and one TMT 10-plex experiments. Bioinformatics analysis showed that Notch signaling, immune response, and protein re-localization processes were uniquely associated with DR, PR, and TAR resistance, respectively. In addition, proteomic signatures related to drug resistance were identified as potential targets of many FDA-approved drugs. Furthermore, we identified potential prognostic proteins with significant effects on overall survival. Representatively, PLXNB2 expression was associated with a highly significant increase in risk, and downregulation of ACOX3 was correlated with a worse overall survival rate. Consequently, our study provides new insights into the proteomic aspects of the distinct mechanisms underlying chemoresistance in breast cancer.

Identification of novel candidate genes implicated in odontogenic potential in the developing mouse tooth germ using transcriptome analysis.

BACKGROUND: In tooth bioengineering for replacement therapy of missing teeth, the utilized cells must possess an inductive signal-forming ability to initiate odontogenesis. This ability is called odontogenic potential. In mice, the odontogenic potential signal is known to be translocated from the epithelium to the mesenchyme at the early bud stage in the developing molar tooth germ. However, the identity of the molecular constituents of this process remains unclear. OBJECTIVE: The purpose of this study is to determine the molecular identity of odontogenic potential and to provide a new perspective in the field of tooth development research. METHODS: In this study, whole transcriptome profiles of the mouse molar tooth germ epithelium and mesenchyme were investigated using the RNA sequencing (RNA-seq) technique. The analyzed transcriptomes corresponded to two developmental stages, embryonic day 11.5 (E11.5) and 14.5 (E14.5), which represent the odontogenic potential shifts. RESULTS: We identified differentially expressed genes (DEGs), which were specifically overexpressed in both the E11.5 epithelium and E14.5 mesenchyme, but not expressed in their respective counterparts. Of the 55 DEGs identified, the top three most expressed transcription factor genes (transcription factor AP-2 beta isoform 3 [TFAP2B], developing brain homeobox protein 2 [DBX2], and insulin gene enhancer protein ISL-1 [ISL1]) and three tooth development-related genes (transcription factor HES-5 [HES5], platelet-derived growth factor D precursor [PDGFD], semaphrin-3 A precursor [SEMA3A]) were selected and validated by quantitative RT-PCR. Using immunofluorescence staining, the TFAP2B protein expression was found to be localized only at the E11.5 epithelium and E14.5 mesenchyme. CONCLUSIONS: Thus, our empirical findings in the present study may provide a new perspective into the characterization of the molecules responsible for the odontogenic potential and may have an implication in the cell-based whole tooth regeneration strategy.

Biochemical activity of magnesium ions on human osteoblast migration.

Magnesium is well known as a biodegradable biomaterial that has been reported to promote bone remodeling in several studies; however, the underlying biological mechanism remains unclear. In the present study, the role of magnesium ions in the migration of U-2 OS cells, which are osteoblast-like cell lines, was investigated. Magnesium treatment did not significantly alter the global transcriptome of U-2 OS cells, but increased the protein expression level of SNAI2, an epithelial-mesenchymal transition (EMT) marker. In addition, it was confirmed that the junctional site localization of Zona-occludens 1 (ZO-1), a representative tight junction protein, was destroyed by magnesium treatment; furthermore, it was determined that cytoplasmic localization increased, and alkaline phosphatase (ALP) activity increased. The obtained results on the mechanism by which magnesium is involved in osteoblast migration, which is important for fracture healing, will contribute to the understanding of the bone-formation process in patients with osteoporosis and musculoskeletal injury.

Is Phylotranscriptomics as Reliable as Phylogenomics?

Phylogenomics, the study of phylogenetic relationships among taxa based on their genome sequences, has emerged as the preferred phylogenetic method because of the wealth of phylogenetic information contained in genome sequences. Genome sequencing, however, can be prohibitively expensive, especially for taxa with huge genomes and when many taxa need sequencing. Consequently, the less costly phylotranscriptomics has seen an increased use in recent years. Phylotranscriptomics reconstructs phylogenies using DNA sequences derived from transcriptomes, which are often orders of magnitude smaller than genomes. However, in the absence of corresponding genome sequences, comparative analyses of transcriptomes can be challenging and it is unclear whether phylotranscriptomics is as reliable as phylogenomics. Here, we respectively compare the phylogenomic and phylotranscriptomic trees of 22 mammals and 15 plants that have both sequenced nuclear genomes and publicly available RNA sequencing data from multiple tissues. We found that phylotranscriptomic analysis can be sensitive to orthologous gene identification. When a rigorous method for identifying orthologs is employed, phylogenomic and phylotranscriptomic trees are virtually identical to each other, regardless of the tissue of origin of the transcriptomes and whether the same tissue is used across species. These findings validate phylotranscriptomics, brighten its prospect, and illustrate the criticality of reliable ortholog detection in such practices.

Identification and sequencing of the gene encoding DNA methyltransferase 3 (DNMT3) from sea cucumber, Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is well known as a traditional tonic food and as a commercially important cultured aquatic species. This species produces saponins, and has a high potential to cope with environmental stress, such as aestivation, organ regeneration, and wound healing. Recently, several studies have shown that cellular reprogramming and the physiological responses of the sea cucumber to environmental changes, including aestivation, are potentially mediated by epigenetic DNA methylation. The DNA methyltransferase (DNMT)1 and DNMT3 genes are independent participants in the maintenance and de novo methylation of specific sequences. Sea urchin (Strongylocentrotus purpuratus) and starfish (Asterina pectinifera), which belong to the same phylum as A. japonicus, have both DNMT1 and DNMT3 genes. However, it was previously reported that DNMT1 is present, but DNMT3 is absent, in A. japonicus. In the present study, we sequenced the full-length cDNA of the A. japonicus DNMT3 gene. The newly sequenced DNMT3 gene comprises three major conserved domains (Pro-Trp-Trp-Pro (PWWP), plant homeodomain (PHD), and S-adenosylmethionine-dependent methyltransferase (AdoMet-MTase)), indicating that the DNMT3 possibly has de novo DNA methylation catalytic activity. Gene structure and phylogenetic analysis showed that sea cucumber DNMT3 is evolutionarily conserved in the Echinodermata. Next, we demonstrated the conservation of DNMT3 gene expression in sea cucumber and starfish belong to same phylum, echinoderm. Using reverse transcription-polymerase chain reaction, sea cucumber DNMT3 mRNA was detected in testis tissue, but not in other tissues tested, including the respiratory tree, muscle, tentacle, intestine, and ovary. This is inconsistent with previous reports, which showed the expression of DNMT3 in ovary, but not in testis of the starfish A. pectinifera, indicating the tissue- and species-specific expression of DNMT3 gene. Although further studies are needed to clarify the epigenetic regulatory mechanisms of DNMT3 and its application to the aquaculture industry, our findings may provide insights into the sea cucumber biology.

Correction: Transcriptome profiling analysis of senescent gingival fibroblasts in response to Fusobacterium nucleatum infection.

[This corrects the article DOI: 10.1371/journal.pone.0188755.].

Genome Sequences of Two Shewanella spp. Isolated from the Gut of the Sea Cucumber Apostichopus japonicus (Selenka, 1867).

In this study, we sequenced the genomes of two Shewanella spp., newly isolated from the gut of the sea cucumber Apostichopus japonicus (Selenka, 1867). The whole-genome sequences reported here will expand the repertoire of genomic information for the members of the genus Shewanella and will provide important insights into their roles within microbial communities.

Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants.

The Japanese sea cucumber (Apostichopus japonicus Selenka 1867) is an economically important species as a source of seafood and ingredient in traditional medicine. It is mainly found off the coasts of northeast Asia. Recently, substantial exploitation and widespread biotic diseases in A. japonicus have generated increasing conservation concern. However, the genomic knowledge base and resources available for researchers to use in managing this natural resource and to establish genetically based breeding systems for sea cucumber aquaculture are still in a nascent stage. A total of 312 Gb of raw sequences were generated using the Illumina HiSeq 2000 platform and assembled to a final size of 0.66 Gb, which is about 80.5% of the estimated genome size (0.82 Gb). We observed nucleotide-level heterozygosity within the assembled genome to be 0.986%. The resulting draft genome assembly comprising 132 607 scaffolds with an N50 value of 10.5 kb contains a total of 21 771 predicted protein-coding genes. We identified 6.6-14.5 million heterozygous single nucleotide polymorphisms in the assembled genome of the three natural color variants (green, red, and black), resulting in an estimated nucleotide diversity of 0.00146. We report the first draft genome of A. japonicus and provide a general overview of the genetic variation in the three major color variants of A. japonicus. These data will help provide a comprehensive view of the genetic, physiological, and evolutionary relationships among color variants in A. japonicus, and will be invaluable resources for sea cucumber genomic research.

Comparative transcriptome analysis of three color variants of the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus Selenka 1867 represents an important resource in biomedical research, traditional medicine, and the seafood industry. Much of the commercial value of A. japonicus is determined by dorsal/ventral color variation (red, green, and black), yet the taxonomic relationships between these color variants are not clearly understood. We performed the first comparative analysis of de novo assembled transcriptome data from three color variants of A. japonicus. Using the Illumina platform, we sequenced nearly 177,596,774 clean reads representing a total of 18.2Gbp of sea cucumber transcriptome. A comparison of over 0.3 million transcript scaffolds against the Uniprot/Swiss-Prot database yielded 8513, 8602, and 8588 positive matches for green, red, and black body color transcriptomes, respectively. Using the Panther gene classification system, we assessed an extensive and diverse set of expressed genes in three color variants and found that (1) among the three color variants of A. japonicus, genes associated with RNA binding protein, oxidoreductase, nucleic acid binding, transferase, and KRAB box transcription factor were most commonly expressed; and (2) the main protein functional classes are differently regulated in all three color variants (extracellular matrix protein and phosphatase for green color, transporter and potassium channel for red color, and G-protein modulator and enzyme modulator for black color). This work will assist in the discovery and annotation of novel genes that play significant morphological and physiological roles in color variants of A. japonicus, and these sequence data will provide a useful set of resources for the rapidly growing sea cucumber aquaculture industry.