Morphological changes in the kidneys of rats with experimental hemorrhagic stroke (intracerebral hemorrhage)

The article presents the results of the study of morphological changes in the kidneys in Wistar rats during the stroke. Hemorrhagic stroke modeling was performed by injecting 0.1-0.2 ml of autoblood through an opening in the temporal bone into the region of the inner capsule of the right hemisphere. To form a hematoma, a stereotaxic destruction was performed. The experimental group consisted of 6 animals, the control group was represented by intact rats (n=3) and rats after trepanation of the temporal bone but without the introduction of autoblood (n=3). The animals were removed from the experiment on days 3 and21. Inthe course of the study, the structural changes in the right and left kidneys were compared to determine the difference in the location of the disorders. According to the results of the histological examination of the medulla, the tendency to reduction of cytological disorders of the epithelium of the tubules of the left kidney was revealed, while in the right kidney the structural disturbances of the tubules of the nephrons and the collecting ducts of the papilla of the kidney were recorded. Based on these data, it is possible to assume renal involvement in rats on day 3 after a stroke and a frequency recovery on day 21. The described histological studies give  a new data on the development of structural changes in renal medulla in rats with hemorrhagic stroke, which can be associated with a violation of electrolyte exchanges at the level of the distal nephron segment

Monte Carlo simulation of signals in digital diaphanoscopy of the maxillary sinuses

Digital diaphanoscopy method has potential to separate normal and pathological conditions of the maxillary sinuses. The entirety of all the features of the investigated area (the presence or absence of pathology, its etiology and morphological features) affects the resulting images of the maxillary sinuses by the digital diaphanoscopy. In this work, the MonteCarlo numerical simulation method was used to determine the patterns of propagation of light radiation in biological tissue. A biologically heterogeneous environment, represented by structures of the skull and maxillary sinuses, as well as pathological changes in them was modelled in the TracePro software

Expression profiling with RNA from formalin-fixed, paraffin-embedded material

<p>Abstract</p> <p>Background</p> <p>Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.</p> <p>Results</p> <p>We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.</p> <p>Conclusion</p> <p>We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.</p

Association of the CpG Methylation Pattern of the Proximal Insulin Gene Promoter with Type 1 Diabetes

The insulin (INS) region is the second most important locus associated with Type 1 Diabetes (T1D). The study of the DNA methylation pattern of the 7 CpGs proximal to the TSS in the INS gene promoter revealed that T1D patients have a lower level of methylation of CpG -19, -135 and -234 (p = 2.10−16) and a higher methylation of CpG -180 than controls, while methylation was comparable for CpG -69, -102, -206. The magnitude of the hypomethylation relative to a control population was 8–15% of the corresponding levels in controls and was correlated in CpGs -19 and -135 (r = 0.77) and CpG -135 and -234 (r = 0.65). 70/485 (14%) of T1D patients had a simultaneous decrease in methylation of CpG -19, -135, -234 versus none in 317 controls. CpG methylation did not correlate with glycated hemoglobin or with T1D duration. The methylation of CpG -69, -102, -180, -206, but not CpG -19, -135, -234 was strongly influenced by the cis-genotype at rs689, a SNP known to show a strong association with T1D. We hypothesize that part of this genetic association could in fact be mediated at the statistical and functional level by the underlying changes in neighboring CpG methylation. Our observation of a CpG-specific, locus-specific methylation pattern, although it can provide an epigenetic biomarker of a multifactorial disease, does not indicate whether the reported epigenetic pattern preexists or follows the establishment of T1D. To explore the effect of chronic hyperglycemia on CpG methylation, we studied non obese patients with type 2 diabetes (T2D) who were found to have decreased CpG-19 methylation versus age-matched controls, similar to T1D (p = 2.10−6) but increased CpG-234 methylation (p = 5.10−8), the opposite of T1D. The causality and natural history of the different epigenetic changes associated with T1D or T2D remain to be determined

Epigenetic Activation of SOX11 in Lymphoid Neoplasms by Histone Modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications

Histone deacetylase controls adult stem cell aging by balancing the expression of polycomb genes and jumonji domain containing 3

Aging is linked to loss of the self-renewal capacity of adult stem cells. Here, we observed that human multipotent stem cells (MSCs) underwent cellular senescence in vitro. Decreased expression of histone deacetylases (HDACs), followed by downregulation of polycomb group genes (PcGs), such as BMI1, EZH2 and SUZ12, and by upregulation of jumonji domain containing 3 (JMJD3), was observed in senescent MSCs. Similarly, HDAC inhibitors induced cellular senescence through downregulation of PcGs and upregulation of JMJD3. Regulation of PcGs was associated with HDAC inhibitor-induced hypophosphorylation of RB, which causes RB to bind to and decrease the transcriptional activity of E2F. JMJD3 expression regulation was dependant on histone acetylation status at its promoter regions. A histone acetyltransferase (HAT) inhibitor prevented replicative senescence of MSCs. These results suggest that HDAC activity might be important for MSC self-renewal by balancing PcGs and JMJD3 expression, which govern cellular senescence by p16INK4A regulation

Identification of Type 1 Diabetes-Associated DNA Methylation Variable Positions That Precede Disease Diagnosis

Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is similar to 50%, implicating roles for genetic and non-genetic factors in the aetiology of this complex autoimmune disease. Although significant progress has been made in elucidating the genetics of T1D in recent years, the non-genetic component has remained poorly defined. We hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology and, thus, performed an epigenome-wide association study (EWAS) for this disease. We generated genome-wide DNA methylation profiles of purified CD14(+) monocytes (an immune effector cell type relevant to T1D pathogenesis) from 15 T1D-discordant MZ twin pairs. This identified 132 different CpG sites at which the direction of the intra-MZ pair DNA methylation difference significantly correlated with the diabetic state, i.e. T1D-associated methylation variable positions (T1D-MVPs). We confirmed these T1D-MVPs display statistically significant intra-MZ pair DNA methylation differences in the expected direction in an independent set of T1D-discordant MZ pairs (P = 0.035). Then, to establish the temporal origins of the T1D-MVPs, we generated two further genome-wide datasets and established that, when compared with controls, T1D-MVPs are enriched in singletons both before (P = 0.001) and at (P = 0.015) disease diagnosis, and also in singletons positive for diabetes-associated autoantibodies but disease-free even after 12 years follow-up (P = 0.0023). Combined, these results suggest that T1D-MVPs arise very early in the etiological process that leads to overt T1D. Our EWAS of T1D represents an important contribution toward understanding the etiological role of epigenetic variation in type 1 diabetes, and it is also the first systematic analysis of the temporal origins of disease-associated epigenetic variation for any human complex disease

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer.

The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer

Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity