The genome of polymorphonuclear neutrophils maintains normal coding sequences

Genetic studies often use genomic DNA from whole blood cells, of which the majority are the polymorphonuclear myeloid cells. Those cells undergo dramatic change of nuclear morphology following cellular differentiation. It remains elusive if the nuclear morphological change accompanies sequence alternations from the intact genome. If such event exists, it will cause a serious problem in using such type of genomic DNA for genetic study as the sequences will not represent the intact genome in the host individuals. Using exome sequencing, we compared the coding regions between neutrophil, which is the major type of polymorphonuclear cells, and CD4+ T cell, which has an intact genome, from the same individual. The results show that exon sequences between the two cell types are essentially the same. The minor differences represented by the missed exons and base changes between the two cell types were validated to be mainly caused by experimental errors. Our study concludes that genomic DNA from whole blood cells can be safely used for genetic studies

Family-specific, novel, deleterious germline variants provide a rich resource to identify genetic predispositions for BRCAx familial breast cancer

BACKGROUND: Genetic predisposition is the primary risk factor for familial breast cancer. For the majority of familial breast cancer, however, the genetic predispositions remain unknown. All newly identified predispositions occur rarely in disease population, and the unknown genetic predispositions are estimated to reach up to total thousands. Family unit is the basic structure of genetics. Because it is an autosomal dominant disease, individuals with a history of familial breast cancer must carry the same genetic predisposition across generations. Therefore, focusing on the cases in lineages of familial breast cancer, rather than pooled cases in disease population, is expected to provide high probability to identify the genetic predisposition for each family. METHODS: In this study, we tested genetic predispositions by analyzing the family-specific variants in familial breast cancer. Using exome sequencing, we analyzed three families and 22 probands with BRCAx (BRCA-negative) familial breast cancer. RESULTS: We observed the presence of family-specific, novel, deleterious germline variants in each family. Of the germline variants identified, many were shared between the disease-affected family members of the same family but not found in different families, which have their own specific variants. Certain variants are putative deleterious genetic predispositions damaging functionally important genes involved in DNA replication and damaging repair, tumor suppression, signal transduction, and phosphorylation. CONCLUSIONS: Our study demonstrates that the predispositions for many BRCAx familial breast cancer families can lie in each disease family. The application of a family-focused approach has the potential to detect many new predispositions

Vitamin D Receptor Deficit Induces Activation of Renin Angiotensin System Via SIRT1 Modulation in Podocytes

Vitamin D receptor (VDR) deficient status has been shown to be associated with the activation of renin angiotensin system (RAS). We hypothesized that lack of VDR would enhance p53 expression in podocytes through down regulation of SIRT1; the former would enhance the transcription of angiotensinogen (Agt) and angiotensinogen II type 1 receptor (AT1R) leading to the activation of RAS. Renal tissues of VDR mutant (M) mice displayed increased expression of p53, Agt, renin, and AT1R. In vitro studies, VDR knockout podocytes not only displayed up regulation p53 but also displayed enhanced expression of Agt, renin and AT1R. VDR deficient podocytes also displayed an increase in mRNA expression for p53, Agt, renin, and AT1R. Interestingly, renal tissues of VDR-M as well as VDR heterozygous (h) mice displayed attenuated expression of deacetylase SIRT1. Renal tissues of VDR-M mice showed acetylation of p53 at lysine (K) 382 residues inferring that enhanced p53 expression in renal tissues could be the result of ongoing acetylation, a consequence of SIRT1 deficient state. Notably, podocytes lacking SIRT1 not only showed acetylation of p53 at lysine (K) 382 residues but also displayed enhanced p53 expression. Either silencing of SIRT1/VDR or treatment with high glucose enhanced podocyte PPAR-y expression, whereas, immunoprecipitation (IP) of their lysates with anti-Retinoid X receptor (RXR) antibody revealed presence of PPAR-y. It appears that either the deficit of SIRT1 has de-repressed expression of PPAR-y or enhanced podocyte expression of PPAR-y (in the absence of VDR) has contributed to the down regulation of SIRT1

New fusion transcripts identified in normal karyotype acute myeloid leukemia

Genetic aberrations contribute to acute myeloid leukemia (AML). However, half of AML cases do not contain the well-known aberrations detectable mostly by cytogenetic analysis, and these cases are classified as normal karyotype AML. Different outcomes of normal karyotype AML suggest that this subgroup of AML could be genetically heterogeneous. But lack of genetic markers makes it difficult to further study this subgroup of AML. Using paired-end RNAseq method, we performed a transcriptome analysis in 45 AML cases including 29 normal karyotype AML, 8 abnormal karyotype AML and 8 AML without karyotype informaiton. Our study identified 134 fusion transcripts, all of which were formed between the partner genes adjacent in the same chromosome and distributed at different frequencies in the AML cases. Seven fusions are exclusively present in normal karyotype AML, and the rest fusions are shared between the normal karyotype AML and abnormal karyotype AML. CIITA, a master regulator of MHC class II gene expression and truncated in B-cell lymphoma and Hodgkin disease, is found to fuse with DEXI in 48% of normal karyotype AML cases. The fusion transcripts formed between adjacent genes highlight the possibility that certain such fusions could be involved in oncological process in AML, and provide a new source to identify genetic markers for normal karyotype AML

Morphine Induces Expression of Platelet-Derived Growth Factor in Human Brain Microvascular Endothelial Cells: Implication for Vascular Permeability

Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF) has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling) and loss-of-function (Egr-1) approaches demonstrated the role of mitogen-activated protein kinases (MAPKs), PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation

Conserved expression of natural antisense transcripts in mammals

BACKGROUND: Recent studies had found thousands of natural antisense transcripts originating from the same genomic loci of protein coding genes but from the opposite strand. It is unclear whether the majority of antisense transcripts are functional or merely transcriptional noise. RESULTS: Using the Affymetrix Exon array with a modified cDNA synthesis protocol that enables genome-wide detection of antisense transcription, we conducted large-scale expression analysis of antisense transcripts in nine corresponding tissues from human, mouse and rat. We detected thousands of antisense transcripts, some of which show tissue-specific expression that could be subjected to further study for their potential function in the corresponding tissues/organs. The expression patterns of many antisense transcripts are conserved across species, suggesting selective pressure on these transcripts. When compared to protein-coding genes, antisense transcripts show a lesser degree of expression conservation. We also found a positive correlation between the sense and antisense expression across tissues. CONCLUSION: Our results suggest that natural antisense transcripts are subjected to selective pressure but to a lesser degree compared to sense transcripts in mammals

Egr-1 expression is up-regulated in HBMECs exposed to morphine.

<p>(<b>A</b>) Time-dependent activation of Egr-1 protein expression by morphine in HBMECs. (<b>B</b>) Representative image of Egr-1 staining in HBMECs treated with morphine. Scale bar = 5 µm. Pretreatment of HBMECs with the inhibitors specific for Erk1/2-U0126 20 µM,JNK-SP600125 20 µM (<b>C</b>) and p38-SB203580 20 µM (<b>D</b>) but not Akt-LY294002 10 µM (<b>C</b>) pathways abrogates morphine-mediated induction of Egr-1. All the data are presented as mean±SD of three individual experiments. ***p<0.001 vs control group; #p<0.05, ###p<0.001 vs morphine group.</p

Effect of morphine on BBB permeability <i>in vitro</i>.

<p>Morphine-mediated increase in BBB permeability was ameliorated in HBMECs pretreated with either naltrexone or PDGF-BB neutralizing antibody. All the data are presented as mean±SD of four individual experiments. *p<0.05 vs control group; #p<0.05 vs morphine-treated group.</p

PDGF-BB induces down regulation of ZO-1 in HBMECs.

<p>(<b>A</b>) Time-dependent reduction of ZO-1 protein expression by morphine in HBMECs. (<b>B</b>) Treatment of cells with either the opioid receptor antagonist (naltrexone) or PDGF-BB neutralizing antibody results in inhibition of ZO-1 expression. All the data are presented as mean±SD of at least three individual experiments. ***p<0.001 vs control group; ###p<0.001 vs morphine-treated group.</p