Long-term expression of glomerular genes in diabetic nephropathy.

Background: Although diabetic nephropathy (DN) is the most common cause for end-stage renal disease in western societies, its pathogenesis still remains largely unclear. A different gene pattern of diabetic and healthy kidney cells is one of the probable explanations. Numerous signalling pathways have emerged as important pathophysiological mechanisms for diabetes-induced renal injury. Methods: Glomerular cells, as podocytes or mesangial cells, are predominantly involved in the development of diabetic renal lesions. While many gene assays concerning DN are performed with whole kidney or renal cortex tissue, we isolated glomeruli from black and tan, brachyuric (BTBR) obese/obese (ob/ob) and wildtype mice at four different timepoints (4, 8, 16 and 24 weeks) and performed an mRNA microarray to identify differentially expressed genes (DEGs). In contrast to many other diabetic mouse models, these homozygous ob/ob leptin-deficient mice develop not only a severe type 2 diabetes, but also diabetic kidney injury with all the clinical and especially histologic features defining human DN. By functional enrichment analysis we were able to investigate biological processes and pathways enriched by the DEGs at different disease stages. Altered expression of nine randomly selected genes was confirmed by quantitative polymerase chain reaction from glomerular RNA. Results: Ob/ob type 2 diabetic mice showed up- and downregulation of genes primarily involved in metabolic processes and pathways, including glucose, lipid, fatty acid, retinol and amino acid metabolism. Members of the CYP4A and ApoB family were found among the top abundant genes. But more interestingly, altered gene loci showed enrichment for processes and pathways linked to angioneogenesis, complement cascades, semaphorin pathways, oxidation and reduction processes and renin secretion. Conclusion: The gene profile of BTBR ob/ob type 2 diabetic mice we conducted in this study can help to identify new key players in molecular pathogenesis of diabetic kidney injury.

Tumor-infiltrating, interleukin-33-producing effector-memory CD8(+) T cells in resected hepatocellular carcinoma prolong patient survival.

UNLABELLED: Interleukin-33 (IL-33), a cytokine with pleiotropic functions, is elevated in serum of patients with hepatocellular carcinoma (HCC). This study investigated the effects of local IL-33 expression in resected HCC on patient survival and on the immunological and molecular tumor microenvironment. Tissue of resected HCCs was stained for hematoxylin and eosin, Masson trichrome, alpha-smooth muscle actin, IL-33, CD8, and IL-13 and analyzed by flow cytometry. Besides histomorphologic evaluation, the immunohistochemical stainings were analyzed for the respective cell numbers separately for tumor area, infiltrative margin, and distant liver stroma. These findings were correlated with clinical data and patient outcome. Further, gene expression of different HCC risk groups was compared using microarrays. In multivariable analysis, infiltration of HCCs by IL-33(+) cells (P = 0.032) and CD8(+) cells (P = 0.014) independently was associated with prolonged patient survival. Flow cytometry demonstrated that cytotoxically active subpopulations of CD8(+) cells, in particular CD8(+) CD62L(-) KLRG1(+) CD107a(+) effector-memory cells, are the main producers of IL-33 in these HCC patients. Using infiltration by IL-33(+) and CD8(+) cells as two separate factors, an HCC immune score was designed and evaluated that stratified patient survival (P = 0.0004). This HCC immune score identified high- and low-risk patients who differ in gene expression profiles (P < 0.001). CONCLUSION: Infiltration of HCCs by IL-33(+) and CD8(+) cells is independently associated with prolonged patient survival. We suggest that this is due to an induction of highly effective, cytotoxically active CD8(+) CD62L(-) KLRG1(+) CD107a(+) effector-memory cells producing IL-33. Based on these two independent factors, we established an HCC immune score that provides risk stratification for HCC patients and can be used in the clinical setting.

Minocycline counter-regulates pro-inflammatory microglia responses in the retina and protects from degeneration.

BACKGROUND: Microglia reactivity is a hallmark of retinal degenerations and overwhelming microglial responses contribute to photoreceptor death. Minocycline, a semi-synthetic tetracycline analog, has potent anti-inflammatory and neuroprotective effects. Here, we investigated how minocycline affects microglia in vitro and studied its immuno-modulatory properties in a mouse model of acute retinal degeneration using bright white light exposure. METHODS: LPS-treated BV-2 microglia were stimulated with 50 µg/ml minocycline for 6 or 24 h, respectively. Pro-inflammatory gene transcription was determined by real-time RT-PCR and nitric oxide (NO) secretion was assessed using the Griess reagent. Caspase 3/7 levels were determined in 661W photoreceptors cultured with microglia-conditioned medium in the absence or presence of minocycline supplementation. BALB/cJ mice received daily intraperitoneal injections of 45 mg/kg minocycline, starting 1 day before exposure to 15.000 lux white light for 1 hour. The effect of minocycline treatment on microglial reactivity was analyzed by immunohistochemical stainings of retinal sections and flat-mounts, and messenger RNA (mRNA) expression of microglia markers was determined using real-time RT-PCR and RNA-sequencing. Optical coherence tomography (OCT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings were used to measure the extent of retinal degeneration and photoreceptor apoptosis. RESULTS: Stimulation of LPS-activated BV-2 microglia with minocycline significantly diminished the transcription of the pro-inflammatory markers CCL2, IL6, and inducible nitric oxide synthase (iNOS). Minocycline also reduced the production of NO and dampened microglial neurotoxicity on 661W photoreceptors. Furthermore, minocycline had direct protective effects on 661W photoreceptors by decreasing caspase 3/7 activity. In mice challenged with white light, injections of minocycline strongly decreased the number of amoeboid alerted microglia in the outer retina and down-regulated the expression of the microglial activation marker translocator protein (18 kDa) (TSPO), CD68, and activated microglia/macrophage whey acidic protein (AMWAP) already 1 day after light exposure. Furthermore, RNA-seq analyses revealed the potential of minocycline to globally counter-regulate pro-inflammatory gene transcription in the light-damaged retina. The severe thinning of the outer retina and the strong induction of photoreceptor apoptosis induced by light challenge were nearly completely prevented by minocycline treatment as indicated by a preserved retinal structure and a low number of apoptotic cells. CONCLUSIONS: Minocycline potently counter-regulates microgliosis and light-induced retinal damage, indicating a promising concept for the treatment of retinal pathologies.

Bacteria-triggered systemic immunity in barley is associated with WRKY and ETHYLENE RESPONSIVE FACTORs but not with salicylic acid.

Leaf-to-leaf systemic immune signaling known as systemic acquired resistance is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to systemic acquired resistance in Arabidopsis (Arabidopsis thaliana), systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid. Instead, we documented a moderate local but not systemic induction of abscisic acid after infection of leaves with Psj. In contrast to salicylic acid or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or abscisic acid triggered systemic immunity to Xtc. RNA sequencing analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest, and quantitative reverse transcription-polymerase chain reaction associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR (ERF)-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors, possibly facilitating transcriptional reprogramming to potentiate immunity.

TFIIS Is Crucial During Early Transcript Elongation for Transcriptional Reprogramming in Response to Heat Stress.

In addition to the stage of transcriptional initiation, the production of mRNAs is regulated during elongation. Accordingly, the synthesis of mRNAs by RNA polymerase II (RNAPII) in the chromatin context is modulated by various transcript elongation factors. TFIIS is an elongation factor that stimulates the transcript cleavage activity of RNAPII to reactivate stalled elongation complexes at barriers to transcription including nucleosomes. Since Arabidopsis tfIIs mutants grow normally under standard conditions, we have exposed them to heat stress (HS), revealing that tfIIs plants are highly sensitive to elevated temperatures. Transcriptomic analyses demonstrate that particularly HS-induced genes are expressed at lower levels in tfIIs than in wildtype. Mapping the distribution of elongating RNAPII uncovered that in tfIIs plants RNAPII accumulates at the +1 nucleosome of genes that are upregulated upon HS. The promoter-proximal RNAPII accumulation in tfIIs under HS conditions conforms to that observed upon inhibition of the RNAPII transcript cleavage activity. Further analysis of the RNAPII accumulation downstream of transcriptional start sites illustrated that RNAPII stalling occurs at +1 nucleosomes that are depleted in the histone variant H2A.Z upon HS. Therefore, assistance of early transcript elongation by TFIIS is required for reprogramming gene expression to establish plant thermotolerance.

Identification of new genes associated with melanoma.

PURPOSE: Repeated failures in melanoma therapy made clear that the molecular mechanisms leading to melanoma are still poorly understood. In this study, we aim to provide a more comprehensive understanding of the transcriptional profiles and signalling pathways associated with melanoma. METHODS: Gene expression was analysed using the Affymetrix Human Genome U133A 2.0 GeneChip arrays. To avoid culture artifacts, we used microdissected fresh frozen material of 18 melanocytic nevi (MN), 20 primary melanomas (PM) and 20 metastatic melanomas (MM). Statistical analysis was performed with Genomatix Chipinspector, Ingenuity™ Software, SPSS Software and Partek Genomic Suite 6.4. Expression levels of selected transcripts were verified by quantitative real-time RT-PCR and immunostaining of a tissue microarray sampling more than 280 cases of MN, PM and MM with known clinical outcome. RESULTS: A total of 284 differentially expressed genes was detected in PM compared with MN and 189 genes in MM compared with PM affecting common cancer pathways such as MAPK-, Wnt- and Notch-signalling. Using principal component analysis, the samples could be grouped according to their histological entity. We identified a panel of novel melanoma-associated markers: frizzled-related protein, an antagonist of Wnt; tranducin-like enhancer of split 1, a transcription factor partner of TCF/LEF-1; CNTN1, an activator of Notch signalling; two Serpin peptidase inhibitors, Serpin B3/B4 and the TGF-ß family member GDF15, the latter with association to MAPK-signalling.

FGFR3 mutation affects cell growth, apoptosis and attachment in keratinocytes.

FGFR3 mutations have recently been identified in several benign epidermal skin lesions such as seborrheic keratosis, epidermal nevus and solar lentigo. The functional consequences of these mutations in human skin are as yet unknown. In this study we analyzed the functional effects of the most common FGFR3 mutation in benign skin tumors, the R248C FGFR3 hotspot mutation, in human HaCaT keratinocytes. The cells were stably transduced with either the R248C or wildtype FGFR3 IIIb cDNA using a retroviral vector system. FGFR3 mutant and wildtype cells showed similar growth rates at subconfluence. However, at confluence FGFR3 mutant keratinocytes revealed a significantly higher cell number than wildtype cells. Furthermore, FGFR3 mutant cells showed significantly lower levels of apoptosis and decreased attachment to fibronectin compared with FGFR3 wildtype cells. Expression of mutant FGFR3 did not alter migration and senescence. Microarray analysis revealed only a few differentially expressed genes between FGFR3 mutant and wildtype keratinocytes. Enhanced phosphorylation of ERK1/2 was observed in confluent R248C mutant HaCaT cells compared with wildtype keratinocytes. Our results suggest that an increased cell number at confluence along with a decreased apoptosis may contribute to the development of acanthotic tumors in FGFR3 mutant skin in vivo.

Stanniocalcin 1 is overexpressed in multipotent mesenchymal stromal cells from acute myeloid leukemia patients.

Objectives: Multipotent mesenchymal stromal cells (MSC) play a pivotal role in the bone marrow (BM) niche. Stanniocalcin 1 (STC1) secreted by MSC has been demonstrated to promote the survival of neoplastic cells and was suggested a marker for minimal residual disease of acute myeloid leukemia (AML). Therefore, we evaluated the expression of STC1 in MSC from AML patients (MSCAML) compared to MSC from healthy donors (MSCHD).Methods: Liquid culture assays of MSCAML and MSCHD were performed to compare expansion capacity. Gene expression profiles of MSCAML vs. MSCHD were established. Secretion of STC1 was tested by ELISA in MSCAML vs. MSCHD and expression of STC1 in AML- vs. HD-BM by immunohistochemistry. In addition, co-cultures of AML cells on MSC were initiated and ultrastructural intercellular communication patterns were investigated. Finally, the effect of blocking STC1 on AML cells was evaluated.Results: MSCAML showed significant decreased expansion capacity compared to MSCHD. Gene analysis revealed marked overexpression of STC1 in MSCAML. ELISA and immunohistochemical findings confirmed this observation. Electron microscopy analysis showed reciprocal stimulation between AML cells and MSC. Blockade of STC1 did not significantly affect AML cell proliferation and apoptosis.Discussion: Characteristics of MSC differ depending on whether they originate from AML patients or from HD. STC1 was mostly overexpressed in MSCAML compared to MSCHD. In vitro blockade of STC1, however, was not associated with AML cell proliferation and apoptosis.Conclusion: Differences in expression levels of glycoproteins from MSCAML compared to MSCHD not necessarily assume that these molecules are niche-relevant in leukemic disease.

SNP dependent modulation of circulating miRNAs from the miR25/93/106 cluster in patients undergoing weight loss.

BACKGROUND: Diet induced weight loss represents an intervention for obesity to prevent associated diseases. However there is considerable inter-individual variation. Single nucleotide polymorphisms (SNPs) and plasma miRNA might be contributing factors. We therefore hypothesized that changes in the miRNA pattern during weight loss depend on the SNP genotype. METHODS: Plasma miRNA profiles from 12 patients were determined before and after a three month weight loss intervention by Illumina sequencing. 46 further patients were analyzed by qPCR. SNP genotypes were determined on the Sequenom iPLEX platform. RESULTS: Samples before and after weight loss were analyzed by miRNA-seq and delta miRNA levels ranked according to p-value. Levels of miRNAs 25, 93 and 106 that are expressed from a common genomic cluster were reduced after weight loss. Those results were substantiated in a qPCR analysis of 46 additional patients. This is in accordance with mouse data showing a functional involvement of this cluster in obesity. Correlation of the changes in miRNA abundance with SNP genotypes revealed a statistical association of all three miRNAs with known obesity susceptibility SNPs. CONCLUSION: Diet induced weight loss leads to SNP dependent modulation of miRNAs from the miR 25/93/106 gene cluster in humans.

Validation of microarray-based resequencing of 93 worldwide mitochondrial genomes.

The human mitochondrial genome consists of a multicopy, circular dsDNA molecule of 16,569 base pairs. It encodes for 13 proteins, two ribosomal genes, and 22 tRNAs that are essential in the generation of cellular ATP by oxidative phosphorylation in eukaryotic cells. Germline mutations in mitochondrial DNA (mtDNA) are an important cause of maternally inherited diseases, while somatic mtDNA mutations may play important roles in aging and cancer. mtDNA polymorphisms are also widely used in population and forensic genetics. Therefore, methods that allow the rapid, inexpensive and accurate sequencing of mtDNA are of great interest. One such method is the Affymetrix GeneChip Human Mitochondrial Resequencing Array 2.0 (MitoChip v.2.0) (Santa Clara, CA). A direct comparison of 93 worldwide mitochondrial genomes sequenced by both the MitoChip and dideoxy terminator sequencing revealed an average call rate of 99.48% and an accuracy of > or =99.98% for the MitoChip. The good performance was achieved by using in-house software for the automated analysis of additional probes on the array that cover the most common haplotypes in the hypervariable regions (HVR). Failure to call a base was associated mostly with the presence of either a run of > or =4 C bases or a sequence variant within 12 bases up- or downstream of that base. A major drawback of the MitoChip is its inability to detect insertions/deletions and its low sensitivity and specificity in the detection of heteroplasmy. However, the vast majority of haplogroup defining polymorphism in the mtDNA phylogeny could be called unambiguously and more rapidly than with conventional sequencing.

Chondroitin sulfate disaccharide stimulates microglia to adopt a novel regulatory phenotype.

A disaccharide degradation product of chondrotin sulfate proteoglycan-disaccharide (CSPG-DS) has been implicated previously in the inhibition of neurodegeneration by influencing microglia activation. In this study, genome-wide microarray analysis was used to identify specific gene expression profiles of CSPG-DS-stimulated BV-2 microglia-like cells. Gene products involved in phagocytosis, detoxification, migration, immune regulation, and antigen presentation were found to be altered significantly. These findings were replicated and compared with IFN-gamma-stimulated primary microglia using real-time quantitative RT-PCR validation. Importantly, a unique transcriptional phenotype with anti-inflammatory and IFN-gamma counter-regulatory properties partially related to alternatively activated macrophages was identified. Using functional cell assays, we found that CSPG-DS-stimulated microglia possess increased phagocytic capacity but lack direct cytotoxic effects such as secretion of NO. Furthermore, conditioned media from CSPG-DS-treated microglia did not diminish the viability or cause apoptosis of cultured photoreceptor cells and partially rescued these cells from IFN-gamma-induced apoptosis. Taken together, our data provide a unique transcript dataset and important in vitro findings about the functional properties of CSPG-DS-activated microglia. These might be starting points to explore the in vivo role of CSPG-DS as a bioactive microglia regulator and its potential, therapeutic application in immune-related, neurodegenerative disorders.

Degradation of D-2-hydroxyglutarate in the presence of isocitrate dehydrogenase mutations.

D-2-Hydroxyglutarate (D-2-HG) is regarded as an oncometabolite. It is found at elevated levels in certain malignancies such as acute myeloid leukaemia and glioma. It is produced by a mutated isocitrate dehydrogenase IDH1/2, a low-affinity/high-capacity enzyme. Its degradation, in contrast, is catalysed by the high-affinity/low-capacity enzyme D-2-hydroxyglutarate dehydrogenase (D2HDH). So far, it has not been proven experimentally that the accumulation of D-2-HG in IDH mutant cells is the result of its insufficient degradation by D2HDH. Therefore, we developed an LC-MS/MS-based enzyme activity assay that measures the temporal drop in substrate and compared this to the expression of D2HDH protein as measured by Western blot. Our data clearly indicate, that the maximum D-2-HG degradation rate by D2HDH is reached in vivo, as vmax is low in comparison to production of D-2-HG by mutant IDH1/2. The latter seems to be limited only by substrate availability. Further, incubation of IDH wild type cells for up to 48 hours with 5 mM D-2-HG did not result in a significant increase in either D2HDH protein abundance or enzyme activity.

RBP2-H1/JARID1B is a transcriptional regulator with a tumor suppressive potential in melanoma cells.

The RBP2-H1/JARID1B nuclear protein belongs to the ARID family of DNA-binding proteins and is a potential tumor suppressor that is lost during melanoma development. As we have recently shown, one physiological function of RBP2-H1/JARID1B is to exert cell cycle control via maintenance of active retinoblastoma protein. We now add new evidence that RBP2-H1/JARID1B can also directly regulate gene transcription in a reporter assay system, either alone or as part of a multimolecular complex together with the developmental transcription factors FOXG1b and PAX9. In melanoma cells, chromatin immunoprecipitation combined with promoter chip analysis (ChIP-on-chip) suggests a direct binding of re-expressed RBP2-H1/JARID1B to a multitude of human regulatory chromosomal elements (promoters, enhancers and introns). Among those, a set of 23 genes, including the melanoma relevant genes CDK6 and JAG-1 could be confirmed by cDNA microarray analyses to be differentially expressed after RBP2-H1/JARID1B re-expression. In contrast, in nonmelanoma HEK 293 cells, RBP2-H1/JARID1B overexpression only evokes a minor transcriptional response in cDNA microarray analyses. Because the transcriptional regulation in melanoma cells is accompanied by an inhibition of proliferation, an increase in caspase activity and a partial cell cycle arrest in G1/0, our data support an anti-tumorigenic role of RBP2-H1/JARID1B in melanocytic cells.

Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte protein (MOG) in female Dark Agouti (DA) rats is a chronic demyelinating animal model of multiple sclerosis (MS). To identify new candidate molecules involved in the evolution or repair of EAE-lesions we used Affymetrix oligonucleotide microarrays to compare the spinal cord transcriptome at the peak of EAE, during remission and at the first relapse with healthy DA rats. METHODS: Untreated DA rats and DA rats immunised with MOG protein were sacrificed at defined time points. Total RNA was isolated from spinal cord tissue and used for hybridization of Affymetrix rat genome arrays RG U34 A-C. Selected expression values were confirmed by RealTime PCR. Adult neural stem cells were incubated with recombinant secretory leukocyte protease inhibitor (SLPI). Proliferation was assessed by BrdU incorporation, cyclin D1 and HES1 expression by RealTime PCR, cell differentiation by immunofluorescence analysis and I kappa B alpha degradation by Western blot. RESULTS: Among approximately 26,000 transcripts studied more than 1,100 were differentially regulated. Focussing on functional themes, we noticed a sustained downregulation of most of the transcripts of the cholesterol biosynthesis pathway. Furthermore, we found new candidate genes possibly contributing to regenerative processes in the spinal cord. Twelve transcripts were solely upregulated in the recovery phase, including genes not previously associated with repair processes. Expression of SLPI was upregulated more than hundredfold during EAE attack. Using immunohistochemistry, SLPI was identified in macrophages, activated microglia, neuronal cells and astrocytes. Incubation of adult neural stem cells (NSC) with recombinant SLPI resulted in an increase of cell proliferation and of differentiation towards oligodendrocytes. These processes were paralleled by an upregulation of the cell-cycle promotor cyclin D1 and a suppression of the cell differentiation regulator HES1. Finally, SLPI prevented the degradation of I kappa B alpha, which may explain the suppression of the cell differentiation inhibitor HES1 suggesting a possible mechanism of oligodendroglial differentiation. CONCLUSION: We identified novel features of gene expression in the CNS during EAE, in particular the suppression of genes of cholesterol biosynthesis and a strong upregulation of SLPI, a gene which is for the first time associated with autoimmune inflammation. The capacity of SLPI to increase proliferation of adult NSC and of oligodendroglial differentiation suggests a novel role for SLPI in the promotion of tissue repair, beyond its known functions in the prevention of tissue damages by protease inhibition damage and modulation of inflammatory reactions.

An integrated workflow for analysis of ChIP-chip data.

Although ChIP-chip is a powerful tool for genome-wide discovery of transcription factor target genes, the steps involving raw data analysis, identification of promoters, and correlation with binding sites are still laborious processes. Therefore, we report an integrated workflow for the analysis of promoter tiling arrays with the Genomatix ChipInspector system. We compare this tool with open-source software packages to identify PU.1 regulated genes in mouse macrophages. Our results suggest that ChipInspector data analysis, comparative genomics for binding site prediction, and pathway/network modeling significantly facilitate and enhance whole-genome promoter profiling to reveal in vivo sites of transcription factor-DNA interactions.

Differential gene expression involved in oxidative stress response caused by triethylene glycol dimethacrylate.

Triethylene glycol dimethacrylate (TEGDMA) is a comonomer that is released from dental resin-based materials into hydrophilic solvents. The compound reduces cell vitality, and causes genotoxicity in mammalian cells in vitro. Here, we used gene expression profiling, combined with pathway analysis tools, to identify the molecular events associated with TEGDMA cytotoxicity in human fibroblasts using Affymetrix HG-U133A 2.0 GeneChip arrays. Increased ROS production and a cell cycle delay caused by 3mm TEGDMA after a 6h exposure were related to a cell response at the transcriptional level. The predominant biological processes associated with the genes that were differentially expressed in untreated and treated cell cultures included oxidative stress, cellular growth, proliferation and morphology, cell death, gene expression as well as DNA replication and repair. The most significantly upregulated genes were GEM (17-fold), KLHL24, DDIT4, TGIF, DUSP5 and ATF3, which are all related to the regulation of the cell structure, stress response, and cell proliferation. TXNIP was the most downregulated transcript (five-fold), whose gene product regulates the cellular redox balance. The downregulation of NRG1, ASPM, FBXO5, and PLK2 is linked to the regulation of cell proliferation and cell structure. The underlying mechanisms of the up- and downregulation of genes seem to be activated by the production of ROS, and the related regulation of the cellular redox balance disturbed in the presence of TEGDMA appears to be of the utmost importance. The coordinated induction of genes coding for oxidative stress response and antioxidant proteins is a critical mechanism of protection against TEGDMA-induced cell damage.

Gene expression profiling of melanocytes following Q-Switched Ruby laser irradiation.

BACKGROUND: The Q-switched Ruby laser (QSRL) is used for the treatment of pigmented lesions. The influence of QSRL treatment on gene expression of nontransformed primary melanocytes has not been addressed in vitro. OBJECTIVE: We investigated the gene expression profile of melanocytes following QSRL irradiation. METHODS: Primary melanocytes were irradiated with the QSRL (694 nm). Early and late transcriptional effects were analyzed using the Affymetrix gene array platform. RESULTS: Laser irradiation of melanocytes had minor effects on mRNA expression. We found only 31 out of 14,500 genes which were at least twofold up- or downregulated. The differential expression of heme oxygenase 1 and galanin in QSRL-treated melanocytes was additionally confirmed by real-time RT-PCR. Analysis of a selection of 36 genes which are known to be associated with malignant melanoma development and progression revealed no significantly aberrant expression in the QSRL-treated melanocytes. CONCLUSION: Our study shows that QSRL treatment of primary melanocytes in vitro does not cause major alterations of global gene expression and particularly of genes associated with malignant melanoma. However, since QSRL treatment may have different effects on gene expression of melanocytic cells in vivo, further studies are required to evaluate QSRL treatment of (nevo-) melanocytic lesions.

Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes.

Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs.

Detecting early signs of heat and drought stress in Phoenix dactylifera (date palm).

Plants adapt to the environment by either long-term genome evolution or by acclimatization processes where the cellular processes and metabolism of the plant are adjusted within the existing potential in the genome. Here we studied the adaptation strategies in date palm, Phoenix dactylifera, under mild heat, drought and combined heat and drought by transcriptomic and metabolomic profiling. In transcriptomics data, combined heat and drought resembled heat response, whereas in metabolomics data it was more similar to drought. In both conditions, soluble carbohydrates, such as fucose, and glucose derivatives, were increased, suggesting a switch to carbohydrate metabolism and cell wall biogenesis. This result is consistent with the evidence from transcriptomics and cis-motif analysis. In addition, transcriptomics data showed transcriptional activation of genes related to reactive oxygen species in all three conditions (drought, heat, and combined heat and drought), suggesting increased activity of enzymatic antioxidant systems in cytosol, chloroplast and peroxisome. Finally, the genes that were differentially expressed in heat and combined heat and drought stresses were significantly enriched for circadian and diurnal rhythm motifs, suggesting new stress avoidance strategies.

A new fate mapping system reveals context-dependent random or clonal expansion of microglia.

Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.