Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming.

Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr-/- mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3-/-/Ldlr-/- mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.

Microbiome Influences Prenatal and Adult Microglia in a Sex-Specific Manner.

Microglia are embryonically seeded macrophages that contribute to brain development, homeostasis, and pathologies. It is thus essential to decipher how microglial properties are temporally regulated by intrinsic and extrinsic factors, such as sexual identity and the microbiome. Here, we found that microglia undergo differentiation phases, discernable by transcriptomic signatures and chromatin accessibility landscapes, which can diverge in adult males and females. Remarkably, the absence of microbiome in germ-free mice had a time and sexually dimorphic impact both prenatally and postnatally: microglia were more profoundly perturbed in male embryos and female adults. Antibiotic treatment of adult mice triggered sexually biased microglial responses revealing both acute and long-term effects of microbiota depletion. Finally, human fetal microglia exhibited significant overlap with the murine transcriptomic signature. Our study shows that microglia respond to environmental challenges in a sex- and time-dependent manner from prenatal stages, with major implications for our understanding of microglial contributions to health and disease.

The tongue microbiome of young patients with chronic kidney disease and their healthy mothers.

OBJECTIVES: Oral microbiome plays a crucial role in the incidence and development of oral diseases. An altered intestinal microbiome has been reported in adults with chronic kidney disease (CKD). This study aimed to characterize the tongue microbiome of young patients with CKD compared to their healthy mothers to identify the influence of CKD-associated factors on resilient tongue ecosystem. MATERIAL AND METHODS: Thirty patients with CKD (mean age, 14.2 years; 16 males and 14 females) and generalized gingivitis were included in the study. Swabs of the posterior tongue were collected from the patients and 21 mothers (mean age 40.8 years). Next-generation sequencing of 16S rDNA genes was employed to quantitatively characterize microbial communities. RESULTS: The bacterial communities were similar in terms of richness and diversity between patients and mothers (p > 0.05). In patients with CKD, 5 core phyla, 20 core genera, and 12 core species were identified. CONCLUSIONS: The tongue microbiome of the study participants showed no relevant CKD-associated differences compared to their mothers and appears to be a highly preserved niche in the oral cavity. Differences observed in the abundance of individual species in this study could be attributed to the age rather than CKD, even after a mean disease duration of 11 years. CLINICAL RELEVANCE: CKD and its associated metabolic changes appear to have no detectable impact on the resilient tongue microbiome observed in young patients.

Integrated genomic surveillance enables tracing of person-to-person SARS-CoV-2 transmission chains during community transmission and reveals extensive onward transmission of travel-imported infections, Germany, June to July 2021.

BackgroundTracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective.AimWe demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters.MethodsGenomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021.ResultsCluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf.ConclusionIGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.

Combined analysis of gut microbiota, diet and PNPLA3 polymorphism in biopsy-proven non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is a global health burden. Risk factors for disease severity include older age, increased body mass index (BMI), diabetes, genetic variants, dietary factors and gut microbiota alterations. However, the interdependence of these factors and their individual impact on disease severity remain unknown. METHODS: In this cross-sectional study, we performed 16S gene sequencing using fecal samples, collected dietary intake, PNPLA3 gene variants and clinical and liver histology parameters in a well-described cohort of 180 NAFLD patients. Principal component analyses were used for dimensionality reduction of dietary and microbiota data. Simple and multiple stepwise ordinal regression analyses were performed. RESULTS: Complete data were available for 57 NAFLD patients. In the simple regression analysis, features associated with the metabolic syndrome had the highest importance regarding liver disease severity. In the multiple regression analysis, BMI was the most important factor associated with the fibrosis stage (OR per kg/m2 : 1.23, 95% CI 1.10-1.37, P < .001). The PNPLA3 risk allele had the strongest association with the histological grade of steatosis (OR 5.32, 95% CI 1.56-18.11, P = .007), followed by specific dietary patterns. Low abundances of Faecalibacterium, Bacteroides and Prevotella and high abundances of Gemmiger were associated with the degree of inflammation, ballooning and stages of fibrosis, even after taking other cofactors into account. CONCLUSIONS: BMI had the strongest association with histological fibrosis, but PNPLA3 gene variants, gut bacterial features and dietary factors were all associated with different histology features, which underscore the multifactorial pathogenesis of NAFLD.

RAL GTPases mediate multiple myeloma cell survival and are activated independently of oncogenic RAS.

Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.

Phenotyping non-alcoholic fatty liver disease by the gut microbiota: Ready for prime time?

BACKGROUND AND AIM: Several studies observed alterations in the gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD). However, analyzed patient populations and methods strongly differ among these studies. The aim of this study was to prove the reproducibility of published results and to provide a detailed overview of all findings in our NAFLD cohort using next generation sequencing methods. METHODS: The individual taxonomic microbiota composition of fecal samples from 90 NAFLD patients and 21 healthy controls was analyzed using 16S rRNA gene sequencing. Study participants were grouped according to their disease stage and compared regarding their gut microbiota composition. Studies were identified from PubMed listed publications, and the results were compared with the findings in our cohort. RESULTS: Results from 13 identified studies were compared with our data. A decreased abundance of the Bacteroidetes and Ruminococcaceae as well as an increased abundance of Lactobacillaceae and Veillonellaceae and Dorea were the most frequently reported changes among NAFLD patients in 4/13, 5/13, 4/13, 2/13, and 3/13 studies, respectively. Even though these alterations in the gut microbiota composition were also observed in our patient cohort, the majority of published differences could not be reproduced, neither in our own nor in other NAFLD cohort studies. CONCLUSION: Despite repeatedly reproduced abundance patterns of specific bacteria, the heterogeneous study results did not reveal a consistent disease specific gut microbiota signature. Further prospective studies with homogenous patient cohorts and standardized methods are necessary to phenotype NAFLD by the gut microbiota.

Nimodipine fosters remyelination in a mouse model of multiple sclerosis and induces microglia-specific apoptosis.

Despite continuous interest in multiple sclerosis (MS) research, there is still a lack of neuroprotective strategies, because the main focus has remained on modulating the immune response. Here we performed in-depth analysis of neurodegeneration in experimental autoimmune encephalomyelitis (EAE) and in in vitro studies regarding the effect of the well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated clinical EAE and spinal cord degeneration and promoted remyelination. Surprisingly, we observed calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell-type specific and irrespective of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS and reactive oxygen species levels in EAE. In addition, increased numbers of Olig2+APC+ oligodendrocytes were detected. Overall, nimodipine application seems to generate a favorable environment for regenerative processes and therefore could be a treatment option for MS, because it combines features of immunomodulation with beneficial effects on neuroregeneration.

APOBEC3G-Regulated Host Factors Interfere with Measles Virus Replication: Role of REDD1 and Mammalian TORC1 Inhibition.

We found earlier that ectopic expression of the cytidine deaminase APOBEC3G (A3G) in Vero cells inhibits measles virus (MV), respiratory syncytial virus, and mumps virus, while the mechanism of inhibition remained unclear. A microarray analysis revealed that in A3G-transduced Vero cells, several cellular transcripts were differentially expressed, suggesting that A3G regulates the expression of host factors. One of the most upregulated host cell factors, REDD1 (regulated in development and DNA damage response-1, also called DDIT4), reduced MV replication ∼10-fold upon overexpression in Vero cells. REDD1 is an endogenous inhibitor of mTORC1 (mammalian target of rapamycin complex-1), the central regulator of cellular metabolism. Interestingly, rapamycin reduced the MV replication similarly to REDD1 overexpression, while the combination of both did not lead to further inhibition, suggesting that the same pathway is affected. REDD1 silencing in A3G-expressing Vero cells abolished the inhibitory effect of A3G. In addition, silencing of A3G led to reduced REDD1 expression, confirming that its expression is regulated by A3G. In primary human peripheral blood lymphocytes (PBL), expression of A3G and REDD1 was found to be stimulated by phytohemagglutinin (PHA) and interleukin-2. Small interfering RNA (siRNA)-mediated depletion of A3G in PHA-stimulated PBL reduced REDD1 expression and increased viral titers, which corroborates our findings in Vero cells. Silencing of REDD1 also increased viral titers, confirming the antiviral role of REDD1. Finally, pharmacological inhibition of mTORC1 by rapamycin in PHA-stimulated PBL reduced viral replication to the level found in unstimulated lymphocytes, indicating that mTORC1 activity supports MV replication as a proviral host factor.IMPORTANCE Knowledge about host factors supporting or restricting virus replication is required for a deeper understanding of virus-cell interactions and may eventually provide the basis for therapeutic intervention. This work was undertaken predominantly to explain the mechanism of A3G-mediated inhibition of MV, a negative-strand RNA virus that is not affected by the deaminase activity of A3G acting on single-stranded DNA. We found that A3G regulates the expression of several cellular proteins, which influences the capacity of the host cell to replicate MV. One of these, REDD1, which modulates the cellular metabolism in a central position by regulating the kinase complex mTORC1, was identified as the major cellular factor impairing MV replication. These findings show interesting aspects of the function of A3G and the dependence of the MV replication on the metabolic state of the cell. Interestingly, pharmacological inhibition of mTORC1 can be utilized to inhibit MV replication in Vero cells and primary human peripheral blood lymphocytes.

The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling.

Cell- and tissue-specific actions of glucocorticoids are mediated by the glucocorticoid receptor. Here, we demonstrate that the glucocorticoid receptor (GR) in macrophages is essential for cardiac healing after myocardial infarction. Compared with GRflox (wild-type controls), GRLysMCre mice that lacked GR in myeloid cells showed increased acute mortality as a result of cardiac rupture. Seven days after left coronary artery ligation, GRLysMCre mice exhibited worse cardiac function and adverse remodeling associated with impaired scar formation and angiogenic response to ischemic injury. Inactivation of GR altered the functional differentiation/maturation of monocyte-derived macrophages in the infarcted myocardium. Mechanistically, CD45+/CD11b+/Ly6G-/F4/80+ macrophages isolated from GRLysMCre infarcts showed deregulation of factors that control inflammation, neovascularization, collagen degradation, and scar tissue formation. Moreover, we demonstrate that cardiac fibroblasts sorted from the ischemic myocardium of GRLysMCre mice compared with cells isolated from injured GRflox hearts displayed higher matrix metalloproteinase 2 expression, and we provide evidence that the macrophage GR regulates myofibroblast differentiation in the infarct microenvironment during the early phase of wound healing. In summary, GR signaling in macrophages, playing a crucial role in tissue-repairing mechanisms, could be a potential therapeutic target during wound healing after ischemic myocardial injury.-Galuppo, P., Vettorazzi, S., Hövelmann, J., Scholz, C.-J., Tuckermann, J. P., Bauersachs, J., Fraccarollo, D. The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling.

Explorative results from multistep screening for potential genetic risk loci of Alzheimer's disease in the longitudinal VITA study cohort.

Alzheimer's disease (AD) is a neurodegenerative disorder that preferentially affects individuals of advanced age. Heritability estimates for AD range between 60 and 80%, but only few genetic risk factors have been identified so far. In the present explorative study, we aimed at characterizing the genetic contribution to late-onset AD in participants of the Vienna Transdanube Aging (VITA) longitudinal birth cohort study in a two-step approach. First, we performed a genome-wide screen of pooled DNA samples (n = 588) to identify allele frequency differences between AD patients and non-AD individuals using life-time diagnoses made at the age of 80 (t = 60 months). This analysis suggested a high proportion of brain-expressed genes required for cell adhesion, cell signaling and cell morphogenesis, and also scored in known AD risk genes. In a second step, we confirmed associations using individual genotypes of top-ranked markers examining AD diagnoses as well as the dimensional scores: FULD and MMSE determined up to the age of 82.5 (t = 90 months). Taken together, our study proposes genes ANKS1B, ENST00000414107, LOC100505811, SLC22A14, QRFPR, ZDHHC8P1, ADAMTS3 and PPFIA1 as possible new candidates involved in the etiology of late-onset AD, with further research being needed to clarify their exact roles.

Differential effects of FTY720 on the B cell compartment in a mouse model of multiple sclerosis.

BACKGROUND: MP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS). METHODS: MP4-immunized mice were treated orally with FTY720 for 30 days at the peak of disease or 50 days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro. RESULTS: FTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs. CONCLUSIONS: The data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE.

High-density preculture of PBMCs restores defective sensitivity of circulating CD8 T cells to virus- and tumor-derived antigens.

Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T cells transiently lose antigen sensitivity when cellular contacts are lost (eg, by entering the circulation). Using the simple and robust protocol for resetting T cells to original reactivity (RESTORE; ie, preculturing PBMCs for 2 days at a high cell density before initiation of antigenic stimulation), we show that CD8 T-cell responses to viral and tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected, if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 protein-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of "restored" CD8 T cells is associated with phosphorylation of proximal T-cell receptor signaling components, and with the upregulation of genes involved in aerobic glycolysis, thereby increasing T-cell functionality. The RESTORE protocol permits a more meaningful monitoring of CD8 memory T-cell responses to viral infections and tumors and vaccination success. Furthermore, when generating T-cell lines for adoptive T-cell therapy, it avoids the loss of those clones, which strictly depend on the primed status conferred by cellular interactions in the tissue context for their initial reactivation by antigen. The data reported in this article have been deposited in the Gene Expression Omnibus database (accession number GSE63430).

The enteric nervous system is a potential autoimmune target in multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.

The regulation of tetraspanin 8 gene expression-A potential new mechanism in the pathogenesis of bipolar disorder.

In a previous study, we identified the single nucleotide polymorphism (SNP) rs4500567, located in the upstream region of tetraspanin 8 (TSPAN8), to be associated with bipolar disorder (BD). Due to its proximal position, the SNP might have an impact on promoter activity, thus on TSPAN8 gene expression. We investigated the impact of rs4500567 on TSPAN8 expression in vitro with luciferase-based promoter assays in human embryonic kidney (HEK293) and neuroblastoma cells (SH-SY5Y), and its effect on expression of downstream associated genes by microarray-based transcriptome analyses. Immunohistochemical localization studies on murine brain slices served to identify possible target regions of altered TSPAN8 expression in the brain. Promoter assays revealed decreased TSPAN8 expression in presence of the minor allele. Transcriptome analyses of TSPAN8-knockdown cells, mirroring the effects of putatively reduced TSPAN8 expression in minor allele carriers, resulted in 231 differentially expressed genes with enrichments of relevant signaling pathways for psychiatric disorders and neuronal development. Finally, we demonstrate Tspan8 abundance in mouse cerebellum and hippocampus. These findings point to a role of TSPAN8 in neuronal function or development. Considering a rather protective effect of the minor allele of rs4500567, our findings reveal a possible novel mechanism that contributes to the development of BD.

Impairment of Wound Healing in Patients With Type 2 Diabetes Mellitus Influences Circulating MicroRNA Patterns via Inflammatory Cytokines.

OBJECTIVE: MicroRNAs (miRNA/miR) are stably present in body fluids and are increasingly explored as disease biomarkers. Here, we investigated influence of impaired wound healing on the plasma miRNA signature and their functional importance in patients with type 2 diabetes mellitus. APPROACH AND RESULTS: miRNA array profiling identified 41 miRNAs significantly deregulated in diabetic controls when compared with patients with diabetes mellitus-associated peripheral arterial disease and chronic wounds. Quantitative real-time polymerase chain reaction validation confirmed decrease in circulating miR-191 and miR-200b levels in type 2 diabetic versus healthy controls. This was reverted in diabetic subjects with associated peripheral arterial disease and chronic wounds, who also exhibited higher circulating C-reactive protein and proinflammatory cytokine levels compared with diabetic controls. miR-191 and miR-200b were significantly correlated with C-reactive protein or cytokine levels in patients with diabetes mellitus. Indeed, proinflammatory stress increased endothelial- or platelet-derived secretion of miR-191 or miR-200b. In addition, dermal cells took up endothelial-derived miR-191 leading to downregulation of the miR-191 target zonula occludens-1. Altered miR-191 expression influenced angiogenesis and migratory capacities of diabetic dermal endothelial cells or fibroblasts, respectively, partly via its target zonula occludens-1. CONCLUSIONS: This study reports that (1) inflammation underlying nonhealing wounds in patients with type 2 diabetes mellitus influences plasma miRNA concentrations and (2) miR-191 modulates cellular migration and angiogenesis via paracrine regulation of zonula occludens-1 to delay the tissue repair process.

Independent natural genetic variation of punishment- versus relief-memory.

A painful event establishes two opponent memories: cues that are associated with pain onset are remembered negatively, whereas cues that coincide with the relief at pain offset acquire positive valence. Such punishment- versus relief-memories are conserved across species, including humans, and the balance between them is critical for adaptive behaviour with respect to pain and trauma. In the fruit fly, Drosophila melanogaster as a study case, we found that both punishment- and relief-memories display natural variation across wild-derived inbred strains, but they do not covary, suggesting a considerable level of dissociation in their genetic effectors. This provokes the question whether there may be heritable inter-individual differences in the balance between these opponent memories in man, with potential psycho-clinical implications.

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status.

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.

DNA methylation mediated control of gene expression is critical for development of crown gall tumors.

Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.

Aneurysm miRNA Signature Differs, Depending on Disease Localization and Morphology.

Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways.