Water deficit response in nodulated soybean roots: a comprehensive transcriptome and translatome network analysis.

BACKGROUND: Soybean establishes a mutualistic interaction with nitrogen-fixing rhizobacteria, acquiring most of its nitrogen requirements through symbiotic nitrogen fixation. This crop is susceptible to water deficit; evidence suggests that its nodulation status-whether it is nodulated or not-can influence how it responds to water deficit. The translational control step of gene expression has proven relevant in plants subjected to water deficit. RESULTS: Here, we analyzed soybean roots' differential responses to water deficit at transcriptional, translational, and mixed (transcriptional + translational) levels. Thus, the transcriptome and translatome of four combined-treated soybean roots were analyzed. We found hormone metabolism-related genes among the differentially expressed genes (DEGs) at the translatome level in nodulated and water-restricted plants. Also, weighted gene co-expression network analysis followed by differential expression analysis identified gene modules associated with nodulation and water deficit conditions. Protein-protein interaction network analysis was performed for subsets of mixed DEGs of the modules associated with the plant responses to nodulation, water deficit, or their combination. CONCLUSIONS: Our research reveals that the stand-out processes and pathways in the before-mentioned plant responses partially differ; terms related to glutathione metabolism and hormone signal transduction (2 C protein phosphatases) were associated with the response to water deficit, terms related to transmembrane transport, response to abscisic acid, pigment metabolic process were associated with the response to nodulation plus water deficit. Still, two processes were common: galactose metabolism and branched-chain amino acid catabolism. A comprehensive analysis of these processes could lead to identifying new sources of tolerance to drought in soybean.

Transcriptomic analysis of N-terminal mutated Trypanosoma cruzi UBP1 knockdown underlines the importance of this RNA-binding protein in parasite development.

BACKGROUND: During its life cycle, the human pathogen Trypanosoma cruzi must quickly adapt to different environments, in which the variation in the gene expression of the regulatory U-rich RNA-binding protein 1 (TcUBP1) plays a crucial role. We have previously demonstrated that the overexpression of TcUBP1 in insect-dwelling epimastigotes orchestrates an RNA regulon to promote differentiation to infective forms. METHODS: In an attempt to generate TcUBP1 knockout parasites by using CRISPR-Cas9 technology, in the present study, we obtained a variant transcript that encodes a protein with 95% overall identity and a modified N-terminal sequence. The expression of this mutant protein, named TcUBP1mut, was notably reduced compared to that of the endogenous form found in normal cells. TcUBP1mut-knockdown epimastigotes exhibited normal growth and differentiation into infective metacyclic trypomastigotes and were capable of infecting mammalian cells. RESULTS: We analyzed the RNA-Seq expression profiles of these parasites and identified 276 up- and 426 downregulated genes with respect to the wildtype control sample. RNA-Seq comparison across distinct developmental stages revealed that the transcriptomic profile of these TcUBP1mut-knockdown epimastigotes significantly differs not only from that of epimastigotes in the stationary phase but also from the gene expression landscape characteristic of infective forms. This is both contrary to and consistent with the results of our recent study involving TcUBP1-overexpressing cells. CONCLUSION: Together, our findings demonstrate that the genes exhibiting opposite changes under overexpression and knockdown conditions unveil key mRNA targets regulated by TcUBP1. These mostly encompass transcripts that encode for trypomastigote-specific surface glycoproteins and ribosomal proteins, supporting a role for TcUBP1 in determining the molecular characteristics of the infective stage.

Differential effects of coverslip-induced hypoxia and cobalt chloride mimetic hypoxia on cellular stress, metabolism, and nuclear structure.

Hypoxia has profound effects on cell physiology, both in normal or pathological settings like cancer. In this study, we asked whether a variant of coverslip-induced hypoxia that recapitulates the conditions found in the tumor microenvironment would elicit similar cellular responses compared to the well established model of cobalt chloride-induced hypoxia. Comparable levels of nuclear HIF-1α were observed after 24 h of coverslip-induced hypoxia or cobalt chloride treatment in CAL-27 oral squamous carcinoma cells. However, cellular stress levels assessed by reactive oxygen species production and lipid droplet accumulation were markedly increased in coverslip-induced hypoxia compared to cobalt chloride treatment. Conversely, mitochondrial ATP production sharply decreased after coverslip-induced hypoxia but was preserved in the presence of cobalt chloride. Coverslip-induced hypoxia also had profound effects in nuclear organization, assessed by changes in nuclear dry mass distribution, whereas these effects were much less marked after cobalt chloride treatment. Taken together, our results show that coverslip-induced hypoxia effects on cell physiology and structure are more pronounced than mimetic hypoxia induced by cobalt chloride treatment. Considering also the simplicity of coverslip-induced hypoxia, our results therefore underscore the usefulness of this method to recapitulate in vitro the effects of hypoxic microenvironments encountered by cells in vivo.

Uncovering a multitude of stage-specific splice variants and putative protein isoforms generated along mouse spermatogenesis.

BACKGROUND: Mammalian testis is a highly complex and heterogeneous tissue. This complexity, which mostly derives from spermatogenic cells, is reflected at the transcriptional level, with the largest number of tissue-specific genes and long noncoding RNAs (lncRNAs) compared to other tissues, and one of the highest rates of alternative splicing. Although it is known that adequate alternative-splicing patterns and stage-specific isoforms are critical for successful spermatogenesis, so far only a very limited number of reports have addressed a detailed study of alternative splicing and isoforms along the different spermatogenic stages. RESULTS: In the present work, using highly purified stage-specific testicular cell populations, we detected 33,002 transcripts expressed throughout mouse spermatogenesis not annotated so far. These include both splice variants of already annotated genes, and of hitherto unannotated genes. Using conservative criteria, we uncovered 13,471 spermatogenic lncRNAs, which reflects the still incomplete annotation of lncRNAs. A distinctive feature of lncRNAs was their lower number of splice variants compared to protein-coding ones, adding to the conclusion that lncRNAs are, in general, less complex than mRNAs. Besides, we identified 2,794 unannotated transcripts with high coding potential (including some arising from yet unannotated genes), many of which encode unnoticed putative testis-specific proteins. Some of the most interesting coding splice variants were chosen, and validated through RT-PCR. Remarkably, the largest number of stage-specific unannotated transcripts are expressed during early meiotic prophase stages, whose study has been scarcely addressed in former transcriptomic analyses. CONCLUSIONS: We detected a high number of yet unannotated genes and alternatively spliced transcripts along mouse spermatogenesis, hence showing that the transcriptomic diversity of the testis is considerably higher than previously reported. This is especially prominent for specific, underrepresented stages such as those of early meiotic prophase, and its unveiling may constitute a step towards the understanding of their key events.

Genomic characterization of Moraxella bovis and Moraxella bovoculi Uruguayan strains isolated from calves with infectious bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.

RNA-seq data exploration after trypanosome RNA-binding protein UBP1 expression is altered by CRISPR-Cas9 gene editing and overexpression.

Previous studies have shown that overexpression of the Trypanosoma cruzi U-rich RNA-binding protein 1 (TcUBP1) in insect-dwelling epimastigotes results in a gene expression pattern resembling that of the infective form of the pathogen. Here, we used CRISPR-Cas9-induced edition of TcUBP1 and full-length protein overexpression in epimastigote cells to monitor transcriptomic changes during the epimastigote-to-metacyclic trypomastigote stage transition of T. cruzi. This dataset includes the bioinformatics analysis of three different RNA-seq samples, each with three biological replicates, showing differential mRNA abundances. The current transcriptome report has the potential to shed light on the quantitative variances in the expression of significant up- or down-regulated mRNAs as a consequence of the levels of the UBP1 protein. Raw data files were deposited at the NCBI Sequence Read Archive - SRA at http://ncbi.nlm.nih.gov/Traces/sra/sra.cgi with accession numbers PRJNA907231 and PRJNA949967.

Estudio de los efectos de un microambiente hipóxico en queratinocitos humanos in vitro y su correlato con alteraciones del microambiente en la patología de liquen plano oral

La hipoxia es un factor fundamental en el proceso de génesis tumoral, así como en patologías precursoras de cáncer, como es el Liquen Plano Oral (LPO). Objetivo: Determinar si es posible establecer una correlación entre las alteraciones que sufren queratinocitos normales en un microambiente hipóxico in vitro y alteraciones que aparecen en los queratinocitos en el epitelio de la mucosa oral en el contexto de la patología LPO. Métodos: Se estudiaron los cambios morfológicos mediante microscopía de contraste de fases, y la detección de marcadores asociados a hipoxia de queratinocitos humanos (HaCaT), como modelo celular oral, en un microambiente hipóxico generado por la variante del método "Hipoxia inducida por cubreobjetos". Resultados: Mediante microscopía confocal se observó la presencia de los marcadores de hipoxia GLUT-1 y aductos de pimonidazol (Hipoxyprobe) en los cultivos celulares de HaCaT expuestos a un microambiente hipóxico. Además, se observó la presencia del marcador GLUT-1 mediante inmunohistoquímica en tejido epitelial humano derivado de biopsias de la patología LPO. Conclusiones: Se estableció una correlación entre las alteraciones detectadas en queratinocitos humanos inducidos a un microambiente hipóxico in vitro y las alteraciones detectadas in vivo en tejido epitelial de la mucosa oral.

A hipóxia é um fator fundamental no processo de gênese tumoral, bem como em patologias precursoras do câncer, como o Líquen Plano Oral (LPO). Objetivo: Determinar se é possível estabelecer uma correlação entre as alterações que os queratinócitos normais sofrem em um microambiente hipóxico in vitro e as alterações que aparecem nos queratinócitos no epitélio da mucosa oral no contexto da patologia do LPO. Métodos: As alterações morfológicas foram estudadas por microscopia de contraste de fase e a detecção de marcadores associados à hipóxia de queratinócitos humanos (HaCaT), como modelo de célula oral, em um microambiente hipóxico gerado pela variante do método "Hipóxia induzida por lamínulas". Resultados: Por microscopia confocal, observou-se a presença dos marcadores de hipóxia GLUT-1 e Hipoxyprobe em culturas de células HaCaT expostas a um microambiente hipóxico. Além disso, a presença do marcador GLUT-1 foi observada por imuno-histoquímica em tecido epitelial humano derivado de biópsias de patologia de LPO. Conclusões: Foi estabelecida uma correlação entre as alterações detectadas em queratinócitos humanos induzidas em um microambiente hipóxico in vitro e as alterações detectadas in vivo no tecido epitelial da mucosa oral.

Hypoxia is a fundamental factor in the process of tumor genesis, as well as in precursor pathologies of cancer, such as Oral Lichen Planus (OLP). Objective: To determine if it is possible to establish a correlation between the alterations that normal keratinocytes suffer in a hypoxic microenvironment in vitro and alterations that appear in the keratinocytes in the epithelium of the oral mucosa in the context of OLP pathology. Methods: Morphological changes were studied by phase contrast microscopy, and the detection of markers associated with hypoxia of human keratinocytes (HaCaT), as an oral cell model, in a hypoxic microenvironment generated by the variant of the method "Hypoxia induced by coverslips". Results: Using confocal microscopy, the presence of hypoxia markers GLUT-1 and Hipoxyprobe was observed in HaCaT cell cultures exposed to a hypoxic microenvironment. In addition, the presence of the GLUT-1 marker was observed by immunohistochemistry in human epithelial tissue derived from biopsies of OLP pathology. Conclusions: A correlation was established between the alterations detected in human keratinocytes induced in a hypoxic microenvironment in vitro and the alterations detected in vivo in epithelial tissue of the oral mucosa.