Significant Transcriptional Alterations Research Articles

Reduced transcriptome analysis in zebrafish uncovers disruptors of spliceosome and ribosome biosynthesis

Abnormal biosynthesis of spliceosomes and ribosomes can lead to their dysfunction, which in turn disrupts protein synthesis and results in various diseases. While genetic factors have been extensively studied, our understanding of how environmental compounds interfere with spliceosome and ribosome biosynthesis remains limited. In the present study, we employed a Reduced Transcriptome Analysis (RTA) approach, integrating large-scale transcriptome data sets of zebrafish and compiling a specific zebrafish gene panel focusing on the spliceosome and ribosome, to elucidate the potential disruptors targeting their biosynthesis. Transcriptomic data sets for 118 environmental substances and 1400 related gene expression profiles were integrated resulting in 513 exposure signatures. Among these substances, several categories including PCB126, transition metals Lanthanum (La) and praseodymium (Pr), heavy metals Cd2+ and AgNO3 and atrazine were highlighted for inducing the significant transcriptional alterations. Furthermore, we found that the transcriptional patterns were distinct between categories, yet overlapping patterns were generally observed within each group. For instance, over 82 % differentially expressed ribosomal genes were shared between La and Pr within the equivalent concentration range. Additionally, transcriptional complexities were also evident across various organs and developmental stages of zebrafish, with notable differences in the inhibition of the transcription of various spliceosome subunits. Overall, our results provide novel insights into the understanding of the adverse effects of environmental compounds, thereby contributing to their environmental risk assessments.

Transcriptome profiling reveals distinct alterations in the B-cell signature and dysregulation of peripheral B-cell subsets in sickle cell anemia patients

Sickle cell anemia (SCA) is characterized by immune system activation and heightened susceptibility to infections. We hypothesized that SCA patients exhibit transcriptional alterations in B-cell-related genes, impacting their peripheral B-cell compartment and leading to dysregulated humoral immunity and increased infection susceptibility. Our objective was to conduct an in silico analysis of whole blood transcriptomes from SCA patients and healthy controls obtained from public repositories. We aimed to identify alterations in the adaptive immune system and validate these findings in our own SCA patient cohort. Bioinformatic analyses unveiled significant transcriptional alterations in B-cell signatures, developmental pathways, and signaling pathways. These results were validated in peripheral blood mononuclear cells from our SCA patient cohort and controls using real-time polymerase chain reaction and flow cytometry. Ninety genes exhibited differential expression, with 70 upregulated and 20 downregulated. Dysregulation in the B-cell compartment of SCA patients was evident, characterized by increased frequencies of immature and naive B-cells, and decreased percentages of memory B-cell subsets compared with healthy controls. Our findings highlight previously unexplored transcriptional and quantitative alterations in peripheral B-cells among SCA patients. Understanding these changes sheds light on the mechanisms contributing to the heightened infection risk in this population. Future studies should delve deeper into these molecular changes to develop targeted interventions and therapeutic strategies aimed at mitigating infection susceptibility in individuals with SCA.

Differentially co-expressed myofibre transcripts associated with abnormal myofibre proportion in chronic obstructive pulmonary disease.

Skeletal muscle dysfunction is a common extrapulmonary manifestation of chronic obstructive pulmonary disease (COPD). Alterations in skeletal muscle myosin heavy chain expression, with reduced type I and increased type II myosin heavy chain expression, are associated with COPD severity when studied in largely male cohorts. The objectives of this study were (1) to define an abnormal myofibre proportion phenotype in both males and females with COPD and (2) to identify transcripts and transcriptional networks associated with abnormal myofibre proportion in COPD. Forty-six participants with COPD were assessed for body composition, strength, endurance and pulmonary function. Skeletal muscle biopsies from the vastus lateralis were assayed for fibre-type distribution and cross-sectional area via immunofluorescence microscopy and RNA-sequenced to generate transcriptome-wide gene expression data. Sex-stratified k-means clustering of type I and IIx/IIax fibre proportions was used to define abnormal myofibre proportion in participants with COPD and contrasted with previously defined criteria. Single transcripts and weighted co-expression network analysis modules were tested for correlation with the abnormal myofibre proportion phenotype. Abnormal myofibre proportion was defined in males with COPD (n=29) as <18% type I and/or >22% type IIx/IIax fibres and in females with COPD (n=17) as <36% type I and/or >12% type IIx/IIax fibres. Half of the participants with COPD were classified as having an abnormal myofibre proportion. Participants with COPD and an abnormal myofibre proportion had lower median handgrip strength (26.1 vs. 34.0kg, P=0.022), 6-min walk distance (300 vs. 353m, P=0.039) and forced expiratory volume in 1s-to-forced vital capacity ratio (0.42 vs. 0.48, P=0.041) compared with participants with COPD and normal myofibre proportions. Twenty-nine transcripts were associated with abnormal myofibre proportions in participants with COPD, with the upregulated NEB, TPM1 and TPM2 genes having the largest fold differences. Co-expression network analysis revealed that two transcript modules were significantly positively associated with the presence of abnormal myofibre proportions. One of these co-expression modules contained genes classically associated with muscle atrophy, as well as transcripts associated with both type I and type II myofibres, and was enriched for genetic loci associated with bone mineral density. Our findings indicate that there are significant transcriptional alterations associated with abnormal myofibre proportions in participants with COPD. Transcripts canonically associated with both type I and type IIa fibres were enriched in a co-expression network associated with abnormal myofibre proportion, suggesting altered transcriptional regulation across multiple fibre types.

Interaction of 17α-ethinylestradiol and methyltestosterone in western mosquitofish (Gambusia affinis) across two generations

The interactions between estrogen and androgen in aquatic animals remain largely unknown. In this study, two generations (F0 and F1) of western mosquitofish (Gambusia affinis) were continuously exposed to 17α-ethinylestradiol (EE2, 10 ng/L), methyltestosterone (MT, 10 ng/L (MTL); 50 ng/L (MTH)), and mixtures (EE2+MTL and EE2+MTH). Various endpoints, including sex ratio (phenotypic and genetic), secondary sex characteristics, gonadal histology, and transcriptional profile of genes, were examined. The results showed that G. affinis exposed to MTH and EE2+MTH had a > 89.7 % of phenotypic males in F1 generation, with 34.5 and 50.0 % of these males originated from genetic females, respectively. Moreover, females from F0 and F1 generations exposed to MTH and EE2+MTH exhibited masculinized anal fins and skeletons. The combined effect of MT and EE2 on most endpoints was dependent on MT. Furthermore, significant transcriptional alterations in certain target genes were observed in both the F0 and F1 generations by EE2 and MT alone and by mixtures, showing some degree of interactions. These findings that the effects of EE2+MTH were primarily on the phenotypic sex of G. affinis in offspring generation suggest that G. affinis under chronic exposure to the binary mixture contaminated water could have sex-biased populations.

Transcriptomic analysis of granulosa cells in patients with endometriosis-related infertility: identification of potential molecular mechanisms

Objective: To investigate the impact of endometriosis on granulosa cell function and elucidate the molecular mechanisms involved. Methods: RNA sequencing, differential expression analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, gene set enrichment analysis, protein-protein interaction analysis, and RT-qPCR were employed to assess the effects of endometriosis on granulosa cell function. Results: The results revealed significant differences in gene expression between the endometriosis and control groups, including genes related to immune regulatory functions and ferroptosis. Hub gene modules and hub genes were identified, including those related to cell cycle and immune and inflammatory pathways. RT-qPCR revealed significant up-regulation of the hub genes CCL3 and IL1B in granulosa cells of patients with endometriosis. Conclusion: The results of RNA sequencing demonstrated that endometriosis induces significant transcriptional alterations in granulosa cells of affected patients. These findings provide important insights into the diagnosis and treatment of endometriosis and highlight the importance of further investigation of CCL3 and IL1B as potential biomarkers for endometriosis.

ACUTE AND SUSTAINED ALTERATIONS TO THE BONE MARROW IMMUNE MICROENVIRONMENT FOLLOWING POLYMICROBIAL INFECTION.

Sepsis is a highly prevalent cause of death in intensive care units. Characterized by severe immune cell derangements, sepsis is often associated with multiorgan dysfunction. For many sepsis survivors, these deficits can persist long after clinical resolution of the underlying infection. Although many studies report on the impact of sepsis on individual immune cell subtypes, a comprehensive analysis of sepsis-induced alterations within and across the immune cell landscape is lacking. In this study, we used single-cell RNA sequencing to assess sepsis-associated transcriptional changes in immune cells isolated from bone marrow at single-cell resolution. We used a high-survival fecal-induced peritonitis sepsis model using Friend leukemia virus B mice. Single-cell RNA sequencing classified 3402 single cells from control subjects into 14 clusters representing long-term hematopoietic stem cell (HSC), short-term HSC, basophil, dendritic cell, eosinophil, erythroblast, erythrocyte, macrophage, neutrophil, natural killer cell, plasma cell, plasmacytoid dendritic cell, pre-B cell, and T memory cell lineages. One day following experimentally induced sepsis, cell type compositions shifted significantly and included notable decreases in HSC and myeloid cell abundance. In addition to proportional cell composition changes, acute sepsis induced significant transcriptional alterations in most immune cell types analyzed-changes that failed to completely resolve 1 month after sepsis. Taken together, we report widespread and persistent transcriptional changes in diverse immune cells in response to polymicrobial infection. This study will serve as a valuable resource for future work investigating acute and/or long-term sepsis-associated immune cell derangements.

Cooperation between KDM6B overexpression and TET2 deficiency in the pathogenesis of chronic myelomonocytic leukemia.

Loss-of-function TET2 mutations are recurrent somatic lesions in chronic myelomonocytic leukemia (CMML). KDM6B encodes a histone demethylase involved in innate immune regulation that is overexpressed in CMML. We conducted genomic and transcriptomic analyses in treatment naïve CMML patients and observed that the patients carrying both TET2 mutations and KDM6B overexpression constituted 18% of the cohort and 42% of patients with TET2 mutations. We therefore hypothesized that KDM6B overexpression cooperated with TET2 deficiency in CMML pathogenesis. We developed a double-lesion mouse model with both aberrations, and discovered that the mice exhibited a more prominent CMML-like phenotype than mice with either Tet2 deficiency or KDM6B overexpression alone. The phenotype includes monocytosis, anemia, splenomegaly, andincreased frequencies and repopulating activity of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). Significant transcriptional alterations were identified in double-lesion mice, which were associated with activation of proinflammatory signals and repression of signals maintaining genome stability. Finally, KDM6B inhibitor reduced BM repopulating activity of double-lesion mice and tumor burden in mice transplanted with BM-HSPCs from CMML patients with TET2 mutations. These data indicate that TET2 deficiency and KDM6B overexpression cooperate in CMML pathogenesis of and that KDM6B could serve as a potential therapeutic target in this disease.

MEDB-62. Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation

Genomic studies in medulloblastoma have identified distinct disease subgroups: wnt/wingless (WNT), sonic hedgehog (SHH), and non-WNT/non-SHH, comprising group 3 and group 4. Alterations in WNT and SHH signalling form the pathogenetic basis for their subgroups, whereas those for non-WNT/non-SHH tumours remain largely elusive. Recent analyses have revealed recurrent in-frame insertions in the E3 ubiquitin ligase adaptor Kelch Repeat and BTB Domain Containing 4 (KBTBD4) in cases of group 3 and group 4 medulloblastoma. Critically, group 3 and 4 tumours with KBTBD4 mutations typically lack other gene-specific alterations, such as MYC amplification, indicating KBTBD4 insertion mutations as the primary genetic driver of these malignancies. Delineating the role of KBTBD4 mutations in medulloblastoma thus offers significant opportunities to understand its pathogenesis and exploit underpinning mechanisms therapeutically, however their function is currently unknown. Here, we show a novel mechanism in cancer pathogenesis whereby indel mutations in KBTBD4 drive its recognition of neo-substrates for degradation. We observe that KBTBD4 mutants promote the recruitment and ubiquitylation of the REST Corepressor (CoREST), which forms a complex to modulate chromatin accessibility and transcriptional programmes. The degradation of CoREST promoted by KBTBD4 mutations diverts epigenetic programmes inducing significant alterations in transcription to promote increased stemness of cancer cells. Transcriptional analysis of >200 human group 3 and 4 medulloblastomas by RNA-seq, highlights the presence of CoREST and stem-like signatures in tumours with KBTBD4 mutations, which extend to a further sub-set of non-mutant tumours, suggesting CoREST alterations as a novel pathogenetic mechanism of wide relevance in group 3 and 4. Our findings uncover KBTBD4 mutation as a novel driver of epigenetic reprogramming in non-WNT/non-SHH medulloblastoma, establishes a novel mode of tumorigenesis through gain-of-function mutations in ubiquitin ligases (neo-substrate recruitment) and identifies both mutant KBTBD4 and CoREST complexes as new druggable targets for improved tumour-specific therapies.

Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation

Medulloblastoma is the most common malignant brain tumour in children. Genomic studies have identified distinct disease subgroups: wnt/wingless (WNT), sonic hedgehog (SHH), and non-WNT/non-SHH, comprising group 3 and group 4. Alterations in WNT and SHH signalling form the pathogenetic basis for their subgroups, whereas those for non-WNT/non-SHH tumours remain largely elusive. Recent analyses have revealed recurrent in-frame insertions in the E3 ubiquitin ligase adaptor Kelch Repeat and BTB Domain Containing 4 (KBTBD4) in cases of group 3/4 medulloblastoma. Critically, group 3/4 tumours with KBTBD4 mutations typically lack other gene-specific alterations, such as MYC amplification, indicating KBTBD4 insertion mutations as the primary genetic driver. Delineating the role of KBTBD4 mutations thus offers significant opportunities to understand tumour pathogenesis and to exploit the underpinning mechanisms therapeutically. Here, we show a novel mechanism in cancer pathogenesis whereby indel mutations in KBTBD4 drive its recognition of neo-substrates for degradation. We observe that KBTBD4 mutants promote the recruitment and ubiquitylation of the REST Corepressor (CoREST), which forms a complex to modulate chromatin accessibility and transcriptional programmes. The degradation of CoREST promoted by KBTBD4 mutation diverts epigenetic programmes inducing significant alterations in transcription to promote increased stemness of cancer cells. Transcriptional analysis of >200 human group 3 and 4 medulloblastomas by RNA-seq, highlights the presence of CoREST and stem-like signatures in tumours with KBTBD4 mutations, which extend to a further sub-set of non-mutant tumours, suggesting CoREST alterations as a novel pathogenetic mechanism of wide relevance in groups 3 and 4. Our findings uncover KBTBD4 mutation as a novel driver of epigenetic reprogramming in non-WNT/non-SHH medulloblastoma, establish a novel mode of tumorigenesis through gain-of-function mutations in ubiquitin ligases (neo-substrate recruitment) and identify both mutant KBTBD4 and CoREST complexes as new druggable targets for improved tumour-specific therapies.

Liver-Dependent Lung Remodeling during Systemic Inflammation Shapes Responses to Secondary Infection.

Systemic duress, such as that elicited by sepsis, burns, or trauma, predisposes patients to secondary pneumonia, demanding better understanding of host pathways influencing this deleterious connection. These pre-existing circumstances are capable of triggering the hepatic acute-phase response (APR), which we previously demonstrated is essential for limiting susceptibility to secondary lung infections. To identify potential mechanisms underlying protection afforded by the lung-liver axis, our studies aimed to evaluate liver-dependent lung reprogramming when a systemic inflammatory challenge precedes pneumonia. Wild-type mice and APR-deficient littermate mice with hepatocyte-specific deletion of STAT3 (hepSTAT3-/-), a transcription factor necessary for full APR initiation, were challenged i.p. with LPS to induce endotoxemia. After 18 h, pneumonia was induced by intratracheal Escherichia coli instillation. Endotoxemia elicited significant transcriptional alterations in the lungs of wild-type and hepSTAT3-/- mice, with nearly 2000 differentially expressed genes between genotypes. The gene signatures revealed exaggerated immune activity in the lungs of hepSTAT3-/- mice, which were compromised in their capacity to launch additional cytokine responses to secondary infection. Proteomics revealed substantial liver-dependent modifications in the airspaces of pneumonic mice, implicating a network of dispatched liver-derived mediators influencing lung homeostasis. These results indicate that after systemic inflammation, liver acute-phase changes dramatically remodel the lungs, resulting in a modified landscape for any stimuli encountered thereafter. Based on the established vulnerability of hepSTAT3-/- mice to secondary lung infections, we believe that intact liver function is critical for maintaining the immunological responsiveness of the lungs.

Biopesticide spinosad induces transcriptional alterations in genes associated with energy production in honey bees (Apis mellifera) at sublethal concentrations.

Bees experience substantial colony losses, which are often associated with pesticides. Besides synthetic insecticides biological compounds such as spinosad are used in agriculture and organic farming against insect pests. However, potential adverse effect at sublethal concentrations to pollinators are poorly known. Here we aim to determine potential adverse outcome pathways of spinosad and to identify molecular effects by investigating transcriptional alterations in the brain of honey bees. We experimentally exposed bees to three sublethal concentrations of 0.05, 0.5 and 5 ng spinosad/bee, and assessed transcriptional alterations of target genes. Additionally, we evaluated whether spinosad-induced transcriptional alterations were influenced by the time of the year. In April, alterations were most pronounced after 24 h exposure, while in June alterations occurred mostly after 48 h. In July, expressional alterations were often lower but the pattern was more similar to that in June than that in April. Down-regulation of genes encoding acetylcholine receptors, enzymes involved in oxidative phosphorylation (cox5a, ndufb7 and cox17), cytochrome P450 dependent monooxygenases (cyp9q1, cyp9q2 and cyp9q3) and insulin-like peptide-1 were among the most significant transcriptional alterations. This suggests adverse effects of spinosad to energy production and metabolism and thus negative consequences on foraging. Together, our study indicates that spinosad causes adverse effects at environmentally realistic concentrations, which may pose a risk to bee populations.

Molecular cloning, characterization and expression analysis of complement components in red sea bream (Pagrus major) after Edwardsiella tarda and red sea bream Iridovirus (RSIV) challenge

The complement system plays an important role in immune regulation and acts as the first line of defense against any pathogenic attack. To comprehend the red sea bream (Pagrus major) immune response, three complement genes, namely, pmC1r, pmMASP and pmC3, belonging to the classical, lectin and alternative complement cascade, respectively, were identified and characterized. pmC1r, pmMASP, and pmC3 were comprised of 2535, 3352, and 5735 base mRNA which encodes 732, 1029 and 1677 aa putative proteins, respectively. Phylogenetically, all the three studied genes clustered with their corresponding homologous clade. Tissue distribution and cellular localization data demonstrated a very high prevalence of all the three genes in the liver. Both bacterial and viral infection resulted in significant transcriptional alterations in all three genes in the liver with respect to their vehicle control counterparts. Specifically, bacterial challenge affected the pmMASP and pmC3 expression, while the viral infection resulted in pmC1r and pmC3 mRNA activation. Altogether, our data demonstrate the ability of pmC1r, pmMASP and pmC3 in bringing about an immune response against any pathogenic encroachment, and thus activating, not only one, but all the three complement pathways, in red sea bream.

Platinum sensitivity of ovarian cancer cells does not influence their ability to induce M2-type macrophage polarization.

Development of platinum resistance in ovarian cancer is mediated by both cancer cells and tumor microenvironment. Activation of epithelial-mesenchymal transition program in cancer cells may lead to enrichment for resistant clones. These processes can be affected by tumor-associated macrophages, a highly plastic population of cells that participate in tumor progression and response to treatment by shaping the microenvironment. We aimed to study how platinum resistance influences the crosstalk between macrophages and ovarian cancer cells. Using cisplatin-sensitive ovarian cancer cell line A2780, we developed and characterized cisplatin-resistant A2780Cis and cisplatin and doxorubicin co-resistant A2780Dox cell lines. Next, we set up an indirect coculture system with THP-1 cell line-derived M0-type-, M1-type- and M2-type-like polarized macrophages. We monitored the expression of genes associated with cellular stemness, multidrug resistance, and epithelial-mesenchymal transition in cancer cells, and expression profile of M1/M2 markers in macrophages. Development of drug resistance in ovarian cancer cell lines was accompanied by increased migration, clonogenicity, and upregulated expression of transcription factors, associated with cellular stemness and epithelial-mesenchymal transition. Upon coculture, we noted that the most relevant changes in gene expression profile occurred in A2780 cells. Moreover, M0- and M1-type macrophages, but not M2-type macrophages, showed significant transcriptional alterations. Our results provide the evidence for bidirectional interplay between cancer cells and macrophages. Independent of platinum resistance status, ovarian cancer cells polarize macrophages toward M2-like type, whereas macrophages induce epithelial-mesenchymal transition and stemness-related gene expression profile in cisplatin-sensitive, but not cisplatin-resistant cancer cells.

Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies.

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain macrophages, we report here a side-by-side comparison of classical cell sort-based transcriptome sequencing and the ‘RiboTag’ method that avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole cell microglia transcriptomes were found to be significantly tainted by artifacts induced by tissue-dissociation, cargo contaminations and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling to assess the accuracy of Cre transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sort-based profiling strategies.

Treponema denticola increases MMP-2 expression and activation in the periodontium via reversible DNA and histone modifications.

Host-derived matrix metalloproteinases (MMPs) and bacterial proteases mediate destruction of extracellular matrices and supporting alveolar bone in periodontitis. The Treponema denticola dentilisin protease induces MMP-2 expression and activation in periodontal ligament (PDL) cells, and dentilisin-mediated activation of pro-MMP-2 is required for cellular fibronectin degradation. Here, we report that T.denticola regulates MMP-2 expression through epigenetic modifications in the periodontium. PDL cells were treated with epigenetic enzyme inhibitors before or after T.denticola challenge. Fibronectin fragmentation, MMP-2 expression, and activation were assessed by immunoblot, zymography, and qRT-PCR, respectively. Chromatin modification enzyme expression in T.denticola-challenged PDL cells and periodontal tissues were evaluated using gene arrays. Several classes of epigenetic enzymes showed significant alterations in transcription in diseased tissue and T.denticola-challenged PDL cells. T.denticola-mediated MMP-2 expression and activation were significantly reduced in PDL cells treated with inhibitors of aurora kinases and histone deacetylases. In contrast, DNA methyltransferase inhibitors had little effect, and inhibitors of histone acetyltransferases, methyltransferases, and demethylases exacerbated T.denticola-mediated MMP-2 expression and activation. Chronic epigenetic changes in periodontal tissues mediated by T.denticola or other oral microbes may contribute to the limited success of conventional treatment of chronic periodontitis and may be amenable to therapeutic reversal.

Transcriptional Alterations of Virulence-Associated Genes in Extended Spectrum Beta-Lactamase (ESBL)-Producing Uropathogenic Escherichia coli during Morphologic Transitions Induced by Ineffective Antibiotics.

It is known that an ineffective antibiotic treatment can induce morphological shifts in uropathogenic Escherichia coli (UPEC) but the virulence properties during these shifts remain to be studied. The present study examines changes in global gene expression patterns and in virulence factor-associated genes in an extended spectrum beta-lactamase (ESBL)-producing UPEC (ESBL019) during the morphologic transitions induced by an ineffective antibiotic and in the presence of human primary bladder epithelial cells. Microarray results showed that the different morphological states of ESBL019 had significant transcriptional alterations of a large number of genes (Transition; 7%, Filamentation; 32%, and Reverted 19% of the entities on the array). All three morphological states of ESBL019 were associated with a decreased energy metabolism, altered iron acquisition systems and altered adhesion expression. In addition, genes associated with LPS synthesis and bacterial motility was also altered in all the morphological states. Furthermore, the transition state induced a significantly higher release of TNF-α from bladder epithelial cells compared to all other morphologies, while the reverted state was unable to induce TNF-α release. Our findings show that the morphological shifts induced by ineffective antibiotics are associated with significant transcriptional virulence alterations in ESBL-producing UPEC, which may affect survival and persistence in the urinary tract.

Effects of antiandrogenic progestins, chlormadinone and cyproterone acetate, and the estrogen 17α-ethinylestradiol (EE2), and their mixtures: Transactivation with human and rainbowfish hormone receptors and transcriptional effects in zebrafish (Danio rerio) eleuthero-embryos

Synthetic progestins act as endocrine disrupters in fish but their risk to the environment is not sufficiently known. Here, we focused on an unexplored antiandrogenic progestin, chlormadinone acetate (CMA), and the antiandrogenic progestin cyproterone acetate (CPA). The aim was to evaluate whether their in vitro interaction with human and rainbowfish (Melanotaenia fluviatilis) sex hormone receptors is similar. Furthermore, we investigated their activity in zebrafish (Danio rerio) eleuthero-embryos.First, we studied agonistic and antagonistic activities of CMA, CPA, and 17α-ethinylestradiol (EE2), in recombinant yeast expressing either the human progesterone (PGR), androgen (AR), or estrogen receptor. The same compounds were also investigated in vitro in a stable transfection cell system expressing rainbowfish nuclear steroid receptors. For human receptors, both progestins exhibited progestogenic, androgenic and antiestrogenic activity with no antiandrogenic or estrogenic activity. In contrast, interactions with rainbowfish receptors showed no progestogenic, but antiandrogenic, antiglucocorticoid, and some antiestrogenic activity. Thus, interaction with and transactivation of human and rainbowfish PGR and AR were distinctly different.Second, we analyzed transcriptional alterations in zebrafish eleuthero‐embryos at 96 and 144h post fertilization after exposure to CPA, CMA, EE2, and binary mixtures of CMA and CPA with EE2, mimicking the use in oral contraceptives. CMA led to slight down-regulation of the ar transcript, while CPA down-regulated ar and pgr transcripts. EE2 exposure resulted in significant transcriptional alterations of several genes, including esr1, pgr, vtg1, cyp19b, and gonadotropins (fshb, lhb). The mixture activity of CMA and EE2 followed the independent action model, while CPA and EE2 mixtures showed additive action in transcriptional alterations.Third, we analyzed the interactions of binary mixtures of CMA and CPA, and of CMA and EE2 for their joint activity in vitro and in eleuthero-embryos. Both mixtures behaved according to the concentration addition model in their in vitro interaction with human and rainbowfish receptors, often showing antagonism. In zebrafish eleuthero-embryos, binary mixtures of CMA and EE2 showed the same expression patterns as EE2 alone, indicating an independent action in vivo.Our study demonstrates that CMA and CPA interact distinctly with human and rainbowfish receptors, suggesting that activities of these and possibly additional environmental steroids determined with yeast expressing human receptors cannot simply be translated to fish. The lack of agonistic activities of both progestins to rainbowfish PGR and AR is the probable reason for the low activity found in zebrafish eleuthero-embryos.

Developmental neurotoxicity of polybrominated diphenyl ethers mixture de71 in Sprague-Dawley rats

ABSTRACTPolybrominated diphenyl ethers (PBDE) are a class of brominated flame retardants that are recognized as global environmental contaminants and a potential adverse health risk. The objective of this study was to evaluate the developmental impacts on rat Sprague-Dawley (SD) pups at postnatal day (PND) 11, 21, 50, 105, and 250 after perinatal exposure to a DE71 mixture. These PNDs corresponded to juveniles, young, and mature adults, respectively. The analysis included histopathological, transcriptional evaluation, and Western blots in both hippocampus and midbrain. There were no marked histopathological changes, but significant transcriptional alterations were observed at PND 21 and 250 in midbrain. These changes occurred in a number of the markers of the cholinergic system, including acetylcholinesterase, muscarinic and nicotinic receptors, and structural gene,s including those of neurofilaments, cell adhesion molecules including N-cadherin and CAMKII, and cytokines. The markers were upregulated at least twofold or greater at PND 21. These biomarkers were predominantly altered in males at low dose (0.3 mg/kg), whereas females were affected only at high concentration (30 mg/kg). At PND 250 both males and females showed downregulation of markers in both intermediate- and high-dose groups. Our results support the findings that in utero and lactational exposure to DE71 mixture leads to transcriptional alterations in midbrain of adult SD rats.

Altered mesenchymal niche cells impede generation of normal hematopoietic progenitor cells in leukemic bone marrow.

Degeneration of normal hematopoietic cells is a shared feature of malignant diseases in the hematopoietic system. Previous studies have shown the exhaustion of hematopoietic progenitor cells (HPCs) in leukemic marrow, whereas hematopoietic stem cells (HSCs) remain functional upon relocation to non-leukemic marrow. However, the underlying cellular mechanisms, especially the specific niche components that are responsible for the degeneration of HPCs, are unknown. In this study, we focused on murine bone mesenchymal stem cells (MSCs) and their supporting function for normal hematopoietic cells in Notch1-induced acute T-cell lymphocytic leukemia (T-ALL) mice. We demonstrate that the proliferative capability and differentiation potential of T-ALL MSCs were impaired due to accelerated cellular senescence. RNA-seq analysis revealed significant transcriptional alterations in leukemic MSCs. After co-cultured with the MSCs from T-ALL mice, a specific inhibitory effect on HPCs was defined, whereas in vivo repopulating potential of normal HSCs was not compromised. Furthermore, osteoprotegerin was identified as a cytokine to improve the function of T-ALL MSCs and to enhance normal HPC output via the p38/ERK pathway. Therefore, this study reveals a novel cellular mechanism underlying the inhibition of HPC generation in T-ALL. Leukemic MSCs may serve as a cellular target for improving normal hematopoietic regeneration therapeutically.

Expression Profile of Angiogenesis Related Genes in Head and Neck Squamous Cell Carcinoma

Objectives: The molecular patterns of tumorigenesis of head and neck squamous cell carcinoma are mostly unknown to date. The concept of this investigation was to expose a genetic fingerprint of angiogenesis in HNSCC with special regard to VEGF as the outstanding mediator of neo-angiogenesis. Material and Methods: A panel of significant transcriptional alterations of VEGF related genes in 83 cancer samples compared to healthy mucosa with microarray technique was established. RT-PCR and immunohistochemistry were performed to confirm the microarray results. Results: In four selected marker genes (VEGFA, RAC2, IL8, PLA2G10) we detected a highly significant transcriptional alteration; three of the genes (VEGFA, RAC2, IL8) showed up-regulation in 88% to 100% of the samples, PLA2G10 down-regulation in 99% of the analyzed tissue samples. Additionally a significant transcription alteration in angio-miRNAs was detected: miR-21 and miR-31 were over-expressed in upto 87% and upto 89% respectively in the analyzed samples. MiR-20a, miR-126, miR-378, miR-17, miR-19a, miR-27b and miR let-7f were down-regulated in 83% to 95% of the specimens. A specific pattern subjected to the different tumor types could not be verified. Conclusion: An expression profile of VEGF related genes was established and selective angio-miRNAs were identified as potential biomarkers on the basis of 83 HNSCC samples. Identification of new relevant oncogenes and insight into the process of tumor vasculogenesis with a highly sensitive method opens new vistas in cancer biology.