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Background Metabolic stress, as negative energy balance on one hand or obesity on the other hand can lead to increased levels of free fatty acids in the plasma and follicular fluid of animals and humans. In an earlier study, we showed that increased oleic acid (OA) concentrations affected the function of cultured bovine granulosa cells (GCs). Here, we focus on genome wide effects of increased OA concentrations. Results Our data showed that 413 genes were affected, of which 197 were down- and 216 up-regulated. Specifically, the expression of FSH-regulated functional key genes, CCND2 , LHCGR , INHA and CYP19A1 and 17-β-estradiol (E2) production were reduced by OA treatment, whereas the expression of the fatty acid transporter CD36 was increased and the morphology of the cells was changed due to lipid droplet accumulation. Bioinformatic analysis revealed that associated pathways of the putative upstream regulators “FSH” and “Cg (choriogonadotropin)” were inhibited and activated, respectively. Down-regulated genes are over-represented in GO terms “reproductive structure/system development”, “ovulation cycle process”, and “(positive) regulation of gonadotropin secretion”, whereas up-regulated genes are involved in “circulatory system development”, “vasculature development”, “angiogenesis” or “extracellular matrix/structure organization”. Conclusions From these data we conclude that besides inhibiting GC functionality, increased OA levels seemingly promote angiogenesis and tissue remodelling, thus suggestively initiating a premature fulliculo-luteal transition. In vivo this may lead to impeded folliculogenesis and ovulation, and cause sub-fertility.

In both humans and mice, obesity is often associated with peripheral and central inflammation, which can lead to diabetes, dysregulation of the stress response, changes in affective behavior, and memory impairment. The DU6 polygenic mouse line was selected over more than 180 generations for a high body mass. Unlike other mouse lines, DU6 mice do not develop diabetes despite significant obesity. We performed a series of behavioral tests on male mice because obesity is often associated with cognitive and emotional impairment. DU6 mice showed no differences in spatial memory or anxiety compared to a control mouse line, based on performance in the Y-maze test, novel object recognition task, and elevated plus-maze test, whereas object memory was impaired in DU6 mice. After psychological stress evoked by the elevated plus-maze test, serum corticosterone concentrations were elevated only in the control mouse line, while corticosterone concentrations were already high in DU6 mice under non-stressed conditions. This elevation under control conditions was no longer detectable at an advanced age. We investigated the composition of immune cells in the spleen and assessed mitogen-induced T-cell activation in vitro in male DU6 mice. Compared to the control mouse line, DU6 mice exhibited significantly fewer CD4 + and CD8 + T cells, alongside a markedly higher proportion of macrophages and Gr-1 + CD11b + myeloid-derived suppressor cells. T-cell activation following mitogen stimulation was lower in DU6 mice than in the control mouse line. Following psychological stress induced by the elevated plus-maze test, the number of CD4+ T cells increased and the number of macrophages decreased in both mouse lines. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were not detectable in the serum of male mice of both lines, ruling out systemic inflammation. Transcriptomic analysis also revealed no inflammation in the hippocampal tissue, but rather a distinct transcriptional signature in male DU6 mice compared to the controls. We propose that the high number of Gr-1 + CD11b + cells protects DU6 mice against systemic inflammation, diabetes, and behavioral impairment.

Background Previously, we could show that L-lactate affects cultured bovine granulosa cells (GC) in a specific manner driving the cells into an early pre-ovulatory phenotype. Here we studied genome wide effects in L-lactate-treated GC to further elucidate the underlying mechanisms that are responsible for the L-lactate induced transformation. Cultured estrogen producing GC treated either with L-lactate or vehicle control were subjected to mRNA microarray analysis. Results The analysis revealed 487 differentially expressed clusters, representing 461 annotated genes. Of these, 333 (= 318 genes) were identified as up- and 154 (= 143 genes) as down-regulated. As the top up-regulated genes we detected TXNIP , H19 and AHSG as well as our previously established marker transcripts RGS2 and PTX3. The top down-regulated genes included VNN1 , SLC27A2 and GFRA1 , but also MYC and the GC marker transcript CYP19A1 . Pathway analysis with differentially expressed genes indicated “cAMP-mediated signaling” and “Axon guidance signaling” among the most affected pathways. Furthermore, estradiol, progesterone and Vegf were identified as potential upstream regulators. An effector network analysis by IPA provided first hints that processes of “angiogenesis” and “vascularization”, but also “cell movement” appeared to be activated, whereas “organismal death” was predicted to be inhibited. Conclusions Our data clearly show that L-lactate alters gene expression in cultured bovine GC in a broad, but obviously specific manner. Pathway analysis revealed that the mode of L-lactate action in GC initiates angiogenic processes, but also migratory events like cell movement and axonal guidance signaling, thus supporting the transformation of GC into an early luteal phenotype.

Background Nutrition has not only an impact on the general wellbeing of an animal but can also affect reproductive processes. In cattle, feeding regimes can influence the age of puberty onset and alter gonadal development. We analyzed effects of different milk replacer (MR) feeding regimes during rearing on ovarian physiology with specific emphasis on the numbers as well as gene expression characteristics of granulosa cells (GCs) at the age of puberty onset. Two groups of calves received either 10% or 20% of bodyweight MR per day during their first 8 weeks. After weaning, both groups were fed the same mixed ration ad libitum until slaughter at 8 months. Results Animals of the 20% feeding group had a significantly higher body weight, but the proportion of animals having a corpus luteum at the time of slaughter was not different between groups, suggesting a similar onset of puberty. Calves of the 10% group showed a constant GC count regardless of the number of follicles (r = 0.23) whereas in the 20% group increasing numbers of GCs were detected with a higher follicle count (r = 0.71). As a first effort to find a possible molecular explanation for this unexpected limitation of GC numbers in the 10% group, we comparatively analyzed GC transcriptomes in both diet groups. The mRNA microarray analysis revealed a total of 557 differentially expressed genes comparing both groups (fold change > |1.5| and p < 0.05). OAS1X , MX2 and OAS1Z were among the top downregulated genes in the 20% vs. the 10% group, whereas top upregulated genes comprised BOLA and XCL1 . All of these genes are known to be regulated by interferon. Subsequent signaling pathway analysis revealed the involvement of several immune response mechanisms in accordance with a number of interferons as upstream regulators. Conclusions The results indicate that the plane of MR feeding in early life has an impact on the number and physiology of GCs later in life. This might influence the overall reproductive life initiated by the onset of puberty in cattle. In addition, the observed alterations in GCs of calves fed less MR might be a consequence of interferon regulated immunological pathways.

BackgroundMultiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS.MethodsOur study focused on 10 bioinformatically prioritized SNP–gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays.ResultsWe were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from GSDMB and SP140 . We also observed an increased IL7R exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 ( EFCAB13 ), rs1131123 ( HLA-C ), rs10783847 (TSFM), and rs2014886 ( TSFM ) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of TSFM due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short TSFM transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed.ConclusionIn summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS.

Background Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called \"immune reconstitution therapies\" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS. Objectives The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy. Methods B cells were obtained from blood samples of patients with relapsing–remitting MS ( n  = 50), patients with primary progressive MS ( n  = 13) as well as healthy controls ( n  = 28). The patients with relapsing MS received either monthly infusions of natalizumab ( n  = 29) or a pulsed IRT with alemtuzumab ( n  = 15) or cladribine ( n  = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration. Results Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2 , IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction. Conclusions We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers.

Current therapeutic approaches have met limited clinical success for glioblastoma multiforme (GBM). Since GBM harbors genomic alterations in cyclin-dependent kinases (CDKs), targeting these structures with specific inhibitors (CDKis) is promising. Here, we describe the antitumoral potential of selective CDKi on low-passage GBM 2D- and 3D models, cultured as neurospheres (NSCs) or glioma stem-like cells (GSCs). By applying selective CDK4/6i abemaciclib and palbociclib, and the more global CDK1/2/5/9-i dinaciclib, different effects were seen. Abemaciclib and dinaciclib significantly affected viability in 2D- and 3D models with clearly visible changes in morphology. Palbociclib had weaker and cell line-specific effects. Motility and invasion were highly affected. Abemaciclib and dinaciclib additionally induced senescence. Also, mitochondrial dysfunction and generation of mitochondrial reactive oxygen species (ROS) were seen. While autophagy was predominantly visible after abemaciclib treatment, dinaciclib evoked γ-H2AX-positive double-strand breaks that were boosted by radiation. Notably, dual administration of dinaciclib and abemaciclib yielded synergistic effects in most cases, but the simultaneous combination with standard chemotherapeutic agent temozolomide (TMZ) was antagonistic. RNA-based microarray analysis showed that gene expression was significantly altered by dinaciclib: genes involved in cell-cycle regulation (different CDKs and their cyclins, SMC3), mitosis (PLK1, TTK), transcription regulation (IRX3, MEN1), cell migration/division (BCAR1), and E3 ubiquitination ligases (RBBP6, FBXO32) were downregulated, whereas upregulation was seen in genes mediating chemotaxis (CXCL8, IL6, CCL2), and DNA-damage or stress (EGR1, ARC, GADD45A/B). In a long-term experiment, resistance development was seen in 1/5 cases treated with dinaciclib, but this could be prevented by abemaciclib. Vice versa, adding TMZ abrogated therapeutic effects of dinaciclib and growth was comparable to controls. With this comprehensive analysis, we confirm the therapeutic activity of selective CDKi in GBM. In addition to the careful selection of individual drugs, the timing of each combination partner needs to be considered to prevent resistance.

Increased life expectancy in industrialized countries is causing an increased incidence of osteoporosis and the need for bioactive bone implants. The integration of implants can be improved physically, but mainly by chemical modifications of the material surface. It was recognized that amino-group-containing coatings improved cell attachment and intracellular signaling. The aim of this study was to determine the role of the amino group density in this positive cell behavior by developing controlled amino-rich nanolayers. This work used covalent grafting of polymer-based nanocoatings with different amino group densities. Titanium coated with the positively-charged trimethoxysilylpropyl modified poly(ethyleneimine) (Ti-TMS-PEI), which mostly improved cell area after 30 min, possessed the highest amino group density with an N/C of 32%. Interestingly, changes in adhesion-related genes on Ti-TMS-PEI could be seen after 4 h. The mRNA microarray data showed a premature transition of the MG-63 cells into the beginning differentiation phase after 24 h indicating Ti-TMS-PEI as a supportive factor for osseointegration. This amino-rich nanolayer also induced higher bovine serum albumin protein adsorption and caused the cells to migrate slower on the surface after a more extended period of cell settlement as an indication of a better surface anchorage. In conclusion, the cell spreading on amine-based nanocoatings correlated well with the amino group density (N/C).

MicroRNAs (miRNAs) are small non-coding RNA molecules acting as post-transcriptional regulators of gene expression. They are involved in many biological processes, and their dysregulation is implicated in various diseases, including multiple sclerosis (MS). Interferon-beta (IFN-beta) is widely used as a first-line immunomodulatory treatment of MS patients. Here, we present the first longitudinal study on the miRNA expression changes in response to IFN-beta therapy. Peripheral blood mononuclear cells (PBMC) were obtained before treatment initiation as well as after two days, four days, and one month, from patients with clinically isolated syndrome (CIS) and patients with relapsing-remitting MS (RRMS). We measured the expression of 651 mature miRNAs and about 19,000 mRNAs in parallel using real-time PCR arrays and Affymetrix microarrays. We observed that the up-regulation of IFN-beta-responsive genes is accompanied by a down-regulation of several miRNAs, including members of the mir-29 family. These differentially expressed miRNAs were found to be associated with apoptotic processes and IFN feedback loops. A network of miRNA-mRNA target interactions was constructed by integrating the information from different databases. Our results suggest that miRNA-mediated regulation plays an important role in the mechanisms of action of IFN-beta, not only in the treatment of MS but also in normal immune responses. miRNA expression levels in the blood may serve as a biomarker of the biological effects of IFN-beta therapy that may predict individual disease activity and progression.