Transcriptome analysis of the Clostridioides difficile response to a specific lactobacilli probiotic formulation: explanations for its mechanisms of action.

AIMS: Clostridioides difficile infections (CDI) are a major cause of morbidity and mortality in hospitalized patients. A probiotic formulation (Bio-K+) comprised of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacti. rhamnosus CLR2 strains have been shown to reduce the incidence of CDI and antibiotic-associated diarrhea (AAD). This research aims to therefore elucidate the mechanism of action of the three probiotic strained against C. difficile R20291, independently of the acidification of the environment. . METHODS AND RESULTS: Antitoxin activity was evaluated using ELISA method and the expression of C. difficile genes was evaluated using transcriptomic analysis in co-culture assays conducted in a bioreactor allowing precise control of the pH. The fermentation results demonstrated a decrease for toxin A and many genes directly related to C. difficile virulence were underexpressed in the co-cultures. CONCLUSIONS: The lactobacilli tested could have a role in the motility, the quorum sensing (QS), the survival of the spores, and the germination potential of the spores, which are essential elements for the virulence of C. difficile. .

Leptosphaeria maculans-Brassica napus Battle: A Comparison of Incompatible vs. Compatible Interactions Using Dual RNASeq.

Leptosphaeria maculans causes blackleg disease, which is one of the most destructive diseases of canola (Brassica napus L.). Due to the erosion of the current resistance in B. napus, it is pivotal to introduce new resistant genotypes to the growers. This study evaluated the potential of Rlm7 gene as resistance to its corresponding avirulence AvrLm7 gene is abundant. The Rlm7 line was inoculated with L. maculans isolate with AvrLm7; UMAvr7; and the CRISPR/Cas9 knockout AvrLm7 mutant, umavr7, of the same isolate to cause incompatible and compatible interactions, respectively. Dual RNA-seq showed differential gene expressions in both interactions. High expressions of virulence-related pathogen genes-CAZymes, merops, and effector proteins after 7-dpi in compatible interactions but not in incompatible interaction-confirmed that the pathogen was actively virulent only in compatible interactions. Salicyclic and jasmonic acid biosynthesis and signaling-related genes, defense-related PR1 gene (GSBRNA2T00150001001), and GSBRNA2T00068522001 in the NLR gene family were upregulated starting as early as 1- and 3-dpi in the incompatible interaction and the high upregulation of those genes after 7-dpi in compatible interactions confirmed the early recognition of the pathogen by the host and control it by early activation of host defense mechanisms in the incompatible interaction.

Transcriptomic Dysregulation of Inflammation-Related Genes in Leukocytes of Patients with Gestational Diabetes Mellitus (GDM) during and after Pregnancy: Identifying Potential Biomarkers Relevant to Glycemic Abnormality.

Although the immune system has been implicated in the pathophysiology of gestational diabetes mellitus (GDM) and postpartum abnormal glucose tolerance (AGT), little is known about the transcriptional response of inflammation-related genes linked to metabolic phenotypes of GDM women during and after pregnancy, which may be potential diagnostic classifiers for GDM and biomarkers for predicting AGT. To address these questions, gene expression of IL6, IL8, IL10, IL13, IL18, TNFA, and the nuclear factor κB (NFκB)/RELA transcription factor were quantified in leukocytes of 28 diabetic women at GDM diagnosis (GDM group) and 1-year postpartum (pGDM group: 10 women with AGT and 18 normoglycemic women), using a nested RT-PCR method. Control pregnancies with normal glucose tolerance (NGT group; n = 31) were closely matched for maternal age, gestational age, pre-pregnancy BMI, pregnancy weight, and gestational weight gain. Compared with the NGT group, IL8 was downregulated in the GDM group, and IL13 and RELA were upregulated in the pGDM group, whereas IL6, IL10, and IL18 were upregulated in the GDM and pGDM groups. The TNFA level did not change from pregnancy to postpartum. Associations of some cytokines with glycemic measures were detected in pregnancy (IL6 and RELA) and postpartum (IL10) (p < 0.05). Receiver operating characteristic (ROC) curves showed that IL6, IL8, and IL18, if employed alone, can discriminate GDM patients from NGT individuals at GDM diagnosis, with the area under the ROC curves (AUCs) of 0.844, (95% CI 0.736−0.953), 0.771 (95% CI 0.651−0.890), and 0.714 (95% CI 0.582−0.846), respectively. By the logistic regression method, we also identified a three-gene panel (IL8, IL13, and TNFA) for postpartum AGT prediction. This study demonstrates a different transcriptional response of the studied genes in clinically well-characterized women with GDM at GDM diagnosis and 1-year postpartum, and provides novel transcriptomic biomarkers for future efforts aimed at diagnosing GDM and identifying the high risk of postpartum AGT groups.

Occurrence of albinism during wheat androgenesis is correlated with repression of the key genes required for proper chloroplast biogenesis.

MAIN CONCLUSION: The phenomenon of albinism in wheat androgenesis is linked to the transcriptional repression of specific genes involved in chloroplast biogenesis during the first weeks of in vitro culture. Isolated microspore culture is widely used to accelerate breeding programs and produce new cultivars. However, in cereals and particularly in wheat, the use of this technique is limited due to the high proportion of regenerated albino plantlets. The causes and mechanisms leading to the formation of albino plantlets in wheat remain largely unknown and, to date, no concrete solution has been found to make it possible to overcome this barrier. We performed a molecular study of proplastid-to-chloroplast differentiation within wheat microspore cultures by analyzing the expression of 20 genes specifically involved in chloroplast biogenesis. Their expression levels were compared between two wheat genotypes that exhibit differential capacities to regenerate green plantlets, i.e., Pavon and Paledor, which produce high and low rates of green plants, respectively. We observed that chloroplast biogenesis within wheat microspores was affected as of the very early stages of the androgenesis process. A successful transition from a NEP- to a PEP-dependent transcription during early plastid development was found to be strongly correlated with the formation of green plantlets, while failure of this transition was strongly correlated with the regeneration of albino plantlets. The very low expression of plastid-encoded 16S and 23S rRNAs within plastids of the recalcitrant genotype Paledor suggests a low translation activity in albino plastids. Furthermore, a delay in the activation of the transcription of nuclear encoded key genes like GLK1 related to chloroplast biogenesis was observed in multicellular structures and pro-embryos of the genotype Paledor. These data help to understand the phenomenon of albinism in wheat androgenesis, which appears to be linked to the transcriptional activation of specific genes involved in the initial steps of chloroplast biogenesis that occurs between days 7 and 21 of in vitro culture.

What determines host specificity in hyperspecialized plant parasitic nematodes?

BACKGROUND: In hyperspecialized parasites, the ability to grow on a particular host relies on specific virulence factors called effectors. These excreted proteins are involved in the molecular mechanisms of parasitism and distinguish virulent pathogens from non-virulent related species. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida are major plant-parasitic nematodes developing on numerous solanaceous species including potato. Their close relatives, G. tabacum and G. mexicana are stimulated by potato root diffusate but unable to establish a feeding site on this plant host. RESULTS: RNA sequencing was used to characterize transcriptomic differences among these four Globodera species and to identify genes associated with host specificity. We identified seven transcripts that were unique to PCN species, including a protein involved in ubiquitination. We also found 545 genes that were differentially expressed between PCN and non-PCN species, including 78 genes coding for effector proteins, which represent more than a 6-fold enrichment compared to the whole transcriptome. Gene polymorphism analysis identified 359 homozygous non-synonymous variants showing a strong evidence for selection in PCN species. CONCLUSIONS: Overall, we demonstrated that the determinant of host specificity resides in the regulation of essential effector gene expression that could be under the control of a single or of very few regulatory genes. Such genes are therefore promising targets for the development of novel and more sustainable resistances against potato cyst nematodes.

Comprehensive transcriptome profiling of soybean leaves in response to simulated acid rain.

As a source of edible oil and protein, soybean is a major globally important economic crop; Improving its production has been an important objective of soybean breeding. Acid rain has been shown to influence soybean growth and productivity, with consequent adverse impacts on its production for use by human populations. In this study, RNA sequencing technology was utilized to examine changes in gene expression when soybean was exposed to simulated acid rain (SAR). We sampled soybean leaves at five time intervals (0, 6, 30, 54, 78, and 102 h), and built the cDNA library. In total, 54,175 expression genes were found, including 2016 genes with differential expression. A total of 416 genes were considered, as they were closely related to the response to SAR. Genes related to the regulation of sulfur and nitrogen metabolism, carbohydrate metabolism, photosynthesis, and reactive oxygen species were among those differentially expressed in response to SAR. In this study, we examined the response mechanisms of soybean under SAR exposure. Our findings will improve our understanding of the molecular mechanisms employed by soybean in responding to abiotic stress, and therefore provides important information in developing soybean breeding to improve tolerance to these stresses.

Auxin and light-mediated regulation of growth, morphogenesis, and alkaloid biosynthesis in Crinum x powellii 'Album' callus.

Crinum x powellii 'Album' belongs to the Amaryllidaceae medicinal plant family that produces a range of structurally diverse alkaloids with potential therapeutic properties. The optimal conditions for in vitro tissue growth, morphogenesis, and alkaloid biosynthesis remain unclear. Auxin and light play critical roles in regulating plant growth, development, and alkaloid biosynthesis in several Amaryllidaceae plants. Here, we have succeeded in showing, for the first time, that the combination of auxin and light significantly influence C. x powellii "Album" in vitro tissue growth, survival, and morphogenesis compared to individual treatments. Furthermore, this combination also upregulates the expression of alkaloid biosynthetic genes and led to an increase in the content of certain alkaloids, suggesting a positive impact on the defense and therapeutic potential of the calli. Our findings provide insights into the regulation of genes involved in alkaloid biosynthesis in C. x powellii "Album" callus and underline the potential of auxin and light as tools for enhancing their production in plants. This study provides a foundation for further exploration of C. x powellii "Album" calli as a sustainable source of bioactive alkaloids for pharmaceutical and agricultural applications. Furthermore, this study paves the way to the discovery of the biosynthetic pathway of specialized metabolites from C. x powellii "Album", such as cherylline and lycorine.

Isolation of a novel Lactiplantibacillus plantarum strain resistant to nitrite stress and its transcriptome analysis.

Nitric oxide (NO) is a reactive nitrogen species (RNS) that plays a vital role in regulating inflammatory processes. Under abnormal conditions, excessive NO levels can promote the oxidation of cellular components, which may cause or exacerbate diseases such as hypertension, cardiovascular dysfunction, and inflammatory bowel disease (IBD). Previous studies have shown that reducing NO levels in the lumen can attenuate the clinical symptoms of IBD. Thus, we aimed to identify bacteria that can reduce RNS and that can be used as valuable probiotics. In this study, we isolated bacteria resistant to nitrite stress from human feces and used 16S and whole-genome sequencing to identify them as Lactiplantibacillus plantarum LP7 (LP7). The ability to survive at high nitrite levels and to decrease them was greater in the LP7 strain than in the reference strain L. plantarum ATCC14917 (ATCC14917). To characterize the LP7 genome in more detail, we performed a comparative genome analysis. However, the unique genes that directly confer the ability to withstand nitrite stress were not present in the LP7 genome. Furthermore, we performed transcriptomic analysis of LP7 and ATCC14917 cells treated with nitrite. We found that the expression levels of genes involved in the cell division process were induced in LP7, which showed a more regular rod-shape than ATCC14917. This could explain why LP7 can survive better than ATCC14917 under nitrite stress. Based on its ability to survive better in nitrite stress and decrease nitrite concentration, we suggest that LP7 could be a valuable probiotic strain.

Characterization of Aldosterone-producing Cell Cluster (APCC) at Single-cell Resolution.

CONTEXT: The adrenal cortex consists of zona glomerulosa (ZG), fasciculata (ZF), and reticularis. Aldosterone-producing cell clusters (APCCs) that strongly express aldosterone synthase (CYP11B2) are frequently found in adult adrenals and harbor somatic mutations that are also detected in aldosterone-producing adenomas (APAs). Primary aldosteronism is mainly caused by APAs or idiopathic hyperaldosteronism (IHA). We presume that APCCs are causing IHA and are precursors of APAs. However, the gene expression characteristics and especially the development of APCCs are not well understood. OBJECTIVE: This study aimed to analyze the transcriptome of APCCs at single-cell resolution and infer the developmental trajectory. METHODS: Single-cell RNA sequencing (scRNA-seq) of 2 adult adrenals was performed. RESULTS: Immunohistochemical analyses confirmed the 2 adrenals had APCCs. scRNA-seq data of 2928 adrenal cells were obtained and 1765 adrenocortical cells were identified based on unsupervised clustering and the marker gene expression. The adrenocortical cells were divided into 6 clusters, of which 3 clusters (923 cells) were composed of APCC/ZG cells. By further subclustering, the APCC/ZG cells were divided into 3 clusters (clusters C1, C2, and C3), we finally identified APCC cluster (C3) and ZG cluster (C1). Cluster C2 seemed to be ZG-to-ZF transitional cells. RNA velocity analysis inferred the developmental direction from cluster ZG-cluster-C1 to APCC-cluster-C3. The scRNA-seq additionally revealed that many CYP11B2-positive cells were positive for CYP11B1 and/or CYP17A1, which were essential for cortisol but not for aldosterone production. CONCLUSIONS: Our results revealed the gene expression characteristics of APCC at single-cell resolution and show that some ZG cells remodel to APCC.

Diaryl Urea Derivative Molecule Inhibits Cariogenic Streptococcus mutans by Affecting Exopolysaccharide Synthesis, Stress Response, and Nitrogen Metabolism.

Different small molecules have been developed to target cariogenic bacteria Streptococcus mutans. Based on target-based designing and in silico screening, a novel diaryl urea derivative, 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea (BPU), has previously been found effective in inhibiting the growth of S. mutans. However, the exact mechanism remains unclear. This current study aimed to explore the antimicrobial and antibiofilm effects of BPU on S. mutans and locate key enzymes and biological processes affected by the molecule via in silico molecular docking analysis and transcriptomic profile. Our in vitro results confirmed that BPU was capable of inhibiting planktonic growth as well as biofilm formation of S. mutans. The virtual binding analysis predicted that the molecule had strong binding potentials with vital enzymes (3AIC and 2ZID) involved in extracellular exopolysaccharide (EPS) synthesis. The predicted inhibitive binding was further confirmed by in vitro quantification of EPS, which found a decreased amount of EPS in the biofilms. The transcriptomic profile also found differential expression of genes involved in EPS synthesis. Moreover, the transcriptomic profile implied alterations in stress response and nitrogen metabolism in S. mutans treated with BPU. Examination of differentially expressed genes involved in these biological processes revealed that altered gene expression could contribute to impaired growth, biofilm formation, and competitiveness of S. mutans. In conclusion, the novel diaryl urea derivative BPU can inhibit the virulence of S. mutans by affecting different biological processes and serves as a potent anti-caries agent.

Analyzing differentially expressed genes and pathways of Bex2-deficient mouse lung via RNA-Seq.

Bex2 is well known for its role in the nervous system, and is associated with neurological disorders, but its role in the lung's physiology is still not reported. To elucidate the functional role of Bex2 in the lung, we generated a Bex2 knock-out (KO) mouse model using the CRISPR-Cas9 technology and performed transcriptomic analysis. A total of 652 genes were identified as differentially expressed between Bex2 -/- and Bex2 +/+ mice, out of which 500 were downregulated, while 152 were upregulated genes. Among these DEGs, Ucp1, Myh6, Coxa7a1, Myl3, Ryr2, RNaset2b, Npy, Enob1, Krt5, Myl2, Hba-a2, and Nrob2 are the most prominent genes. Myl2, was the most downregulated gene, followed by Npy, Hba-a2, Rnaset2b, nr0b2, Klra8, and Ucp1. Tcte3, Eno1b, Zfp990, and Pcdha9 were the most upregulated DEGs. According to gene enrichment analysis, PPAR pathway, cardiac muscle contraction, and cytokine-cytokine receptor interaction were the most enriched pathways. Besides, the nuclear factor-κB signaling pathway and hematopoietic cell linage pathways were also enriched. Chronic obstructive pulmonary disease (COPD) is enriched among KEGG disease pathways. RT-qPCR assays confirmed the RNA-Seq results. This study opens a new window toward the biological functions of Bex2 in different systems.

Comparative transcriptome analysis of gene expression patterns on B16F10 melanoma cells under Photobiomodulation of different light modes.

Cutaneous melanoma is one of the aggressive cancers. Recent studies have shown that Photobiomodulation (PBM) can inhibit the proliferation of melanoma cells. However, it is not clear that the effect of PBM light mode on the inhibition of melanoma cells. Herein, we investigated the difference of influence between continuous wave (CW) and Pulse PBM on B16F10 melanoma cells. Our results suggested that Pulse mode had a more significant inhibition on the viability of B16F10 melanoma cells than CW mode under the PBM light parameter of wavelength, dose, and average irradiance at 457 nm, 1.14 J/cm2, and 0.19 mW/cm2. Besides, we revealed the differentially expressed genes of B16F10 melanoma cells under the various treatments of PBM light mode (not PBM treatment, CW mode, and Pulse mode) by RNA sequencing. Together, our data suggested that Pulse-PBM can improve the effect of PBM on cells significantly and there may be different molecular mechanisms between Pulse and CW mode including anti-proliferative and cell necrosis. The study shed new light on investigating the molecular mechanisms of various PBM light modes on B16F10 melanoma cells.

Vitamin D3-Induced Tolerogenic Dendritic Cells Modulate the Transcriptomic Profile of T CD4+ Cells Towards a Functional Hyporesponsiveness.

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most robust approaches due to their immune regulatory properties, which are currently being tested in clinical trials. However, the mechanisms that vitD3-tolDC trigger for the induction of tolerance remain elusive. For this reason, we performed a full phenotypical, functional, and transcriptomic characterization of T cells upon their interaction with autologous, antigen-specific vitD3-tolDC. We observed a strong antigen-specific reduction of T cell proliferation, combined with a decrease in the relative prevalence of TH1 subpopulations and IFN-γ production. The analysis of the transcriptomic profile of T CD4+ cells evidenced a significant down-modulation of genes involved in cell cycle and cell response to mainly pro-inflammatory immune-related stimuli, highlighting the role of JUNB gene as a potential biomarker of these processes. Consequently, our results show the induction of a strong antigen-specific hyporesponsiveness combined with a reduction on the TH1 immune profile of T cells upon their interaction with vitD3-tolDC, which manifests the regulatory properties of these cells and, therefore, their therapeutic potential in the clinic.

Differential gene expression in the condylar cartilage of growing rabbits with temporomandibular joint anterior disk displacement-A transcriptomic study.

Internal derangement (ID) in the temporomandibular joint (TMJ) comprises a group of clinical problems with relatively high prevalence. However, the temporal changes in gene expression of condylar cartilage during continuous ID remain unclear. The aim of the current study is to investigate by microarray analysis, the differentially-expressed gene pattern in condylar cartilage of rabbits with ID from one to eight weeks of ID progression. Histological results (hematoxylin and eosin staining) indicated that abnormal collagen fiber arrangements, fragmentation of fibrils, and inflammatory cell-infiltration were detected from one to four weeks in joint disc specimens, while newly formed vessels, mucoid degeneration, meniscal tears, and the presence of osteoclasts and osteoblasts were observed at later time points. The microarray analysis revealed 6478 genes among all tested transcripts, to have a greater than two-fold expression change compared to controls. The inflammation-associated gene group including ace and il1ß increased rapidly in the early stage of disease and decreased later. In contrast, bone construction-related genes showed low expression levels at first and increased at later period in the ID progression. The current study also found some genes such as hla2g, which have not been previously reported, to be potentially relevant within ID. Our findings provide useful insights into the pathological mechanism of ID in TMJ.

Identification of microRNA-mRNA dysregulations in paroxysmal atrial fibrillation.

BACKGROUND: The molecular mechanisms underlying the early development of atrial fibrillation (AF) remain poorly understood. Emerging evidence suggests that abnormal epigenetic modulation via microRNAs (miRNAs) might be involved in the pathogenesis of paroxysmal AF (pAF). OBJECTIVE: To identify key molecular changes associated with pAF, we conducted state-of-the-art transcriptomic studies to identify the abnormal miRNA-mRNA interactions potentially driving AF development. METHODS: High-quality total RNA including miRNA was isolated from atrial biopsies of age-matched and sex-matched pAF patients and control patients in sinus rhythm (SR; n=4 per group) and used for RNA-sequencing and miRNA microarray. Results were analyzed bioinformatically and validated using quantitative real-time (qRT)-PCR and 3'UTR luciferase reporter assays. RESULTS: 113 genes and 49 miRNAs were differentially expressed (DE) in pAF versus SR patients. Gene ontology analysis revealed that most of the DE genes were involved in the "gonadotropin releasing hormone receptor pathway" and "p53 pathway". Of these DE genes, bioinformatic analyses identified 23 pairs of putative miRNA-mRNA interactions that were altered in pAF (involving 15 miRNAs and 17 mRNAs). Using qRT-PCR and 3'UTR luciferase reporter assays, the interaction between upregulation of miR-199a-5p and downregulation of FKBP5 was confirmed in samples from pAF patients. CONCLUSION: Our combined transcriptomic analysis and miRNA microarray study of atrial samples from pAF patients revealed novel pathways and miRNA-mRNA regulations that may be relevant in the development of pAF. Future studies are required to investigate the potential involvement of the gonadotropin releasing hormone receptor and p53 pathways in AF pathogenesis.

Transcriptomic study on the impact of temporomandibular joint internal derangement in the condylar cartilage of rabbits.

Internal derangement (ID) in the temporomandibular joint (TMJ) compromises a group of clinical problems, and holds a relative high prevalence in populations. However, the temporal genomic change in gene expression of condylar cartilage during continuous ID remains unclear. Here we reported the differentially expressed gene pattern in condylar cartilage of rabbits with ID from 1 to 8 weeks by microarray analysis. The whole genome project was deposited at GenBank under the accession PRJNA278127. The microarray analysis showed that 6478 genes have more than two-fold changes among all the tested transcripts. Many inflammation gene increased rapidly in the early stage while decrease later. On the contrary, the bone construction related genes showed a low level at first and increased at later period in the ID progression. Besides, the current study found some genes such as HLA2G, which had never been reported, might be relevant with ID.