High Density Genetic Maps of Seashore Paspalum Using Genotyping-By-Sequencing and Their Relationship to The Sorghum Bicolor Genome.

As a step towards trait mapping in the halophyte seashore paspalum (Paspalum vaginatum Sw.), we developed an F1 mapping population from a cross between two genetically diverse and heterozygous accessions, 509022 and HI33. Progeny were genotyped using a genotyping-by-sequencing (GBS) approach and sequence reads were analyzed for single nucleotide polymorphisms (SNPs) using the UGbS-Flex pipeline. More markers were identified that segregated in the maternal parent (HA maps) compared to the paternal parent (AH maps), suggesting that 509022 had overall higher levels of heterozygosity than HI33. We also generated maps that consisted of markers that were heterozygous in both parents (HH maps). The AH, HA and HH maps each comprised more than 1000 markers. Markers formed 10 linkage groups, corresponding to the ten seashore paspalum chromosomes. Comparative analyses showed that each seashore paspalum chromosome was syntenic to and highly colinear with a single sorghum chromosome. Four inversions were identified, two of which were sorghum-specific while the other two were likely specific to seashore paspalum. These high-density maps are the first available genetic maps for seashore paspalum. The maps will provide a valuable tool for plant breeders and others in the Paspalum community to identify traits of interest, including salt tolerance

Glucose-induced down regulation of thiamine transporters in the kidney proximal tubular epithelium produces thiamine insufficiency in diabetes

Increased renal clearance of thiamine (vitamin B1) occurs in experimental and clinical diabetes producing thiamine insufficiency mediated by impaired tubular re-uptake and linked to the development of diabetic nephropathy. We studied the mechanism of impaired renal re-uptake of thiamine in diabetes. Expression of thiamine transporter proteins THTR-1 and THTR-2 in normal human kidney sections examined by immunohistochemistry showed intense polarised staining of the apical, luminal membranes in proximal tubules for THTR-1 and THTR-2 of the cortex and uniform, diffuse staining throughout cells of the collecting duct for THTR-1 and THTR-2 of the medulla. Human primary proximal tubule epithelial cells were incubated with low and high glucose concentration, 5 and 26 mmol/l, respectively. In high glucose concentration there was decreased expression of THTR-1 and THTR-2 (transporter mRNA: −76% and −53% respectively, p<0.001; transporter protein −77% and −83% respectively, p<0.05), concomitant with decreased expression of transcription factor specificity protein-1. High glucose concentration also produced a 37% decrease in apical to basolateral transport of thiamine transport across cell monolayers. Intensification of glycemic control corrected increased fractional excretion of thiamine in experimental diabetes. We conclude that glucose-induced decreased expression of thiamine transporters in the tubular epithelium may mediate renal mishandling of thiamine in diabetes. This is a novel mechanism of thiamine insufficiency linked to diabetic nephropathy

Genome-Wide Gene Expression in relation to Age in Large Laboratory Cohorts of \u3ci\u3eDrosophila melanogaster\u3c/i\u3e

Aging is a complex process characterized by a steady decline in an organism’s ability to perform life-sustaining tasks. In the present study, two cages of approximately 12,000 mated Drosophila melanogaster females were used as a source of RNA from individuals sampled frequently as a function of age. A linear model for micro array data method was used for the micro array analysis to adjust for the box effect; it identified 1,581 candidate aging genes.Cluster analyses using a self-organizing map algorithm on the 1,581 significant genes identified gene expression patterns across different ages. Genes involved in immune system function and regulation, chorion assembly and function, and metabolism were all significantly differentially expressed as a function of age. The temporal pattern of data indicated that gene expression related to aging is affected relatively early in life span. In addition, the temporal variance in gene expression in immune function genes was compared to a random set of genes. There was an increase in the variance of gene expression within each cohort, which was not observed in the set of random genes.This observation is compatible with the hypothesis that D. melanogaster immune function genes lose control of gene expression as flies age

miR-27b*, an Oxidative Stress-Responsive microRNA Modulates Nuclear Factor-kB Pathway in RAW 264.7 Cells

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H2O2) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H2O2. These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway

Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex

<p>Abstract</p> <p>Background</p> <p>The reduced folate carrier (<it>RFC1</it>) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine <it>RFC1 </it>gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 <it>RFC1</it>^-/-embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation.</p> <p>Results</p> <p>Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 <it>RFC1</it>^-/-vs. <it>RFC1</it>^+/+embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of <it>RFC1</it>^-/-embryos, while cubilin protein was widely misexpressed.</p> <p>Conclusion</p> <p>Inactivation of <it>RFC1 </it>impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate and other nutrients, lipids and morphogens such as sonic hedgehog (Shh) and retinoids that play critical roles in normal embryogenesis.</p

Mammalian alteration/deficiency in activation 3 (Ada3) is essential for embryonic development and cell cycle progression.

Ada3 protein is an essential component of histone acetyl transferase containing coactivator complexes conserved from yeast to human. We show here that germline deletion of Ada3 in mouse is embryonic lethal, and adenovirus-Cre mediated conditional deletion of Ada3 in Ada3(FL/FL) mouse embryonic fibroblasts leads to a severe proliferation defect which was rescued by ectopic expression of human Ada3. A delay in G(1) to S phase of cell cycle was also seen that was due to accumulation of Cdk inhibitor p27 which was an indirect effect of c-myc gene transcription control by Ada3. We further showed that this defect could be partially reverted by knocking down p27. Additionally, drastic changes in global histone acetylation and changes in global gene expression were observed in microarray analyses upon loss of Ada3. Lastly, formation of abnormal nuclei, mitotic defects and delay in G(2)/M to G(1) transition was seen in Ada3 deleted cells. Taken together, we provide evidence for a critical role of Ada3 in embryogenesis and cell cycle progression as an essential component of HAT complex

Distinct gene expression profiles in different B-cell compartments in human peripheral lymphoid organs

BACKGROUND: There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naïve cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments. RESULTS: We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known. CONCLUSIONS: Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors

Damaging Cardiac and Cancer Genetic Variants in the LVAD Population

Background: Next generation sequencing technology, coupled with population genetic databases, have made broad genetic evaluation relatively inexpensive and widely available. Our objective was to assess the prevalence of potentially damaging cancer and cardiac gene variants in advanced non-ischemic cardiomyopathy patients. Methods: Explanted human heart tissue procured at LVAD placement was obtained from the University of Nebraska Medical Center Heart Tissue Bank. Genomic DNA was isolated from tissues and amplified by PCR using targeted ampliseq primer pools from an inherited disease panel. Individual libraries were amplified by emulsion PCR on Ion Sphere particles and sequencing was performed on a PGM sequencer (Ion torrent) using the Ion 316 chip. The Ion Torrent browser suite was used to map the reads and call the variants. The identified single nucleotide polymorphisms, insertions, and deletions were then annotated and characterized with ANNOVAR. Non-synonymous mutations with a population frequency of less than or equal to 1% were identified and analyzed utilizing an open source integrative genomics viewer. Amino acid substitution effects on protein function were determined by a bioinformatics algorithm. Myocardial recovery was defined as an improvement in EF to greater than 45% at three months post implant. Results: Our sample population included 12 males and 2 females with an average age of 49 and an average EF at presentation of 17%. Damaging cardiac gene variants were present in 11/14 patients. Only 1 of the 11 patients with damaging cardiac gene variants improved their ejection fraction to greater than 45% post LVAD. Two of the 2 patients without mutations improved their ejection fraction to greater than 45%, p-value=.04. Nine of the 14 patients in this population had damaging oncogene mutations. Conclusions: Damaging variants in cancer and cardiac genes are common in end-stage non-ischemic cardiomyopathy patients undergoing LVAD placement. Genetic variation likely contributes to disease progression and cancer risk

High-Throughput Analysis of Lung Immune Cells in a Combined Murine Model of Agriculture Dust-Triggered Airway Inflammation With Rheumatoid Arthritis

Rheumatoid arthritis (RA)-associated lung disease is a leading cause of mortality in RA, yet the mechanisms linking lung disease and RA remain unknown. Using an established murine model of RA-associated lung disease combining collagen-induced arthritis (CIA) with organic dust extract (ODE)-induced airway inflammation, differences among lung immune cell populations were analyzed by single cell RNA-sequencing. Additionally, four lung myeloid-derived immune cell populations including macrophages, monocytes/macrophages, monocytes, and neutrophils were isolated by fluorescence cell sorting and gene expression was determined by NanoString analysis. Unsupervised clustering revealed 14 discrete clusters among Sham, CIA, ODE, and CIA+ODE treatment groups: 3 neutrophils (inflammatory, resident/transitional, autoreactive/suppressor), 5 macrophages (airspace, differentiating/recruited, recruited, resident/interstitial, and proliferative airspace), 2 T-cells (differentiating and effector), and a single cluster each of inflammatory monocytes, dendritic cells, B-cells and natural killer cells. Inflammatory monocytes, autoreactive/suppressor neutrophils, and recruited/differentiating macrophages were predominant with arthritis induction (CIA and CIA+ODE). By specific lung cell isolation, several interferon-related and autoimmune genes were disproportionately expressed among CIA and CIA+ODE (e.g. Oasl1, Oas2, Ifit3, Gbp2, Ifi44, and Zbp1), corresponding to RA and RA-associated lung disease. Monocytic myeloid-derived suppressor cells were reduced, while complement genes (e.g. C1s1 and Cfb) were uniquely increased in CIA+ODE mice across cell populations. Recruited and inflammatory macrophages/monocytes and neutrophils expressing interferon-, autoimmune-, and complement-related genes might contribute towards pro-fibrotic inflammatory lung responses following airborne biohazard exposures in setting of autoimmune arthritis and could be predictive and/or targeted to reduce disease burden

Genome-wide expression profiling reveals transcriptomic variation and perturbed gene networks in androgen-dependent and androgen-independent prostate cancer cells.

Previously, we have developed a unique in vitro LNCaP cell model, which includes androgen-dependent (LNCaP-C33), androgen-independent (LNCaP-C81) and an intermediate phenotype (LNCaP-C51) cell lines resembling the stages of prostate cancer progression to hormone independence. This model is advantageous in overcoming the heterogeneity associated with the prostate cancer up to a certain extent. We characterized and compared the gene expression profiles in LNCaP-C33 (androgen-dependent) and LNCaP-C81 (androgen-independent) cells using Affymetrix GeneChip array analyses. Multiple genes were identified exhibiting differential expression during androgen-independent progression. Among the important genes upregulated in androgen-independent cells were PCDH7, TPTE, TSPY, EPHA3, HGF, MET, EGF, TEM8, etc., whereas many candidate tumor suppressor genes (HTATIP2, CDKN2A, CDKN2B, CDKN1C, TP53, TP73, ICAM1, SOCS1/2, SPRY2, PPP2CA, PPP3CA, etc.) were decreased. Pathway prediction analysis identified important gene networks associated with growth-promoting and apoptotic signaling that were perturbed during androgen-independent progression. Further investigation of one of the genes, PPP2CA, which encodes the catalytic subunit of a serine phosphatase PP2A, a potent tumor suppressor, revealed that its expression was decreased in prostate cancer compared to adjacent normal/benign tissue. Furthermore, the downregulated expression of PPP2CA was significantly correlated with tumor stage and Gleason grade. Future studies on the identified differentially expressed genes and signaling pathways may be helpful in understanding the biology of prostate cancer progression and prove useful in developing novel prognostic biomarkers and therapy for androgen-refractory prostate cancer