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Wang X.,Duke University | Wang P.,The Research Institute for Children | Wang P.,Louisiana State University Health Sciences Center | Sun S.,Duke University | And 3 more authors.
PLoS Genetics | Year: 2012

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (~50%), but at a much lower frequency during vegetative growth (~0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ~90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling. © 2012 Wang et al. Source


McMurtry V.E.,Louisiana State University Health Sciences Center | Gupta R.W.,Childrens Hospital | Tran L.,Pediatrix Medical Group of Louisiana | Blanchard E.E.,Louisiana State University Health Sciences Center | And 4 more authors.
Microbiome | Year: 2015

Background: Necrotizing enterocolitis (NEC) is a devastating neonatal gastrointestinal disease that primarily affects premature infants. It is characterized by bowel inflammation and necrosis. In spite of extensive research, there has been little progress in decreasing the incidence or mortality of NEC over the past three decades. The exact etiology of NEC has not been identified. However, it is believed to result from an inappropriate immune response to gut microbiota. Using 454-pyrosequencing analyses of 16S rRNA genes that were PCR-amplified from stool DNA specimens, we compared the gut microbiota of infants with NEC to matched controls without NEC. The infants with NEC were then categorized into three subgroups based on severity: mild, severe, and lethal. We compared the microbiota among these subgroups and between each severity group and appropriate controls. Results: Bacterial diversity and the relative abundance of Actinobacteria and Clostridia were significantly lower in NEC specimens compared to controls. The absence of Clostridia was significantly associated with NEC. Microbial diversity and Clostridia abundance and prevalence decreased with increasing severity of NEC. Conclusions: Low bacterial diversity in stool specimens may be indicative of NEC and the severity of NEC. The low bacterial diversity, and the lack of Clostridia in lethal specimens, could indicate that the presence of a diverse bacterial population in the gut as well as the presence of taxa such as Clostridia may play a role in attenuating inflammation leading to NEC. © 2015 McMurtry et al.; licensee BioMed Central. Source


Chakraborty S.,University of New Orleans | Chakraborty S.,Central Connecticut State University | Cai Y.,University of New Orleans | Cai Y.,The Research Institute for Children | Tarr M.A.,University of New Orleans
Proteomics | Year: 2014

Low-density lipoprotein (LDL) is a major cholesterol carrier in human blood. Oxidations of apolipoprotein B-100 (apo B-100, LDL protein) could be proatherogenic and play critical roles in early stages of plaque formation in the arterial wall. The structure of apo B-100 is still poorly understood, partially due to its size (550 KDa, 4563 amino acids). To gain an insight into LDL structure, we mapped the regions of apo B-100 in human LDL that were prone to oxidation using peroxynitrite and hypochlorite as probes. In this study, LDL was incubated with various concentrations of peroxynitrite and sodium hypochlorite in bicarbonate buffer. The LDL protein apo B-100 was delipidated, denatured, alkylated, and subjected to tryptic digestion. Tryptic peptides were analyzed employing LC-MS/MS. Database search was performed against the apo B-100 database (SwissProt accession #P04114) using "SEQUEST" algorithm to identify peroxynitrite and hypochlorite-mediated oxidations markers nitrotyrosine, nitrotryptophan, hydroxy-tryptophan, and 3-chlorotyrosine. Several site-specific oxidations were identified in apo B-100 after treatment of intact LDL particles with the oxidants. We hypothesize that these regions could be accessible to oxidant and critical for early events in atherosclerotic plaque deposition. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Abbaraju N.V.,University of New Orleans | Cai Y.,University of New Orleans | Cai Y.,The Research Institute for Children | Rees B.B.,University of New Orleans
Proteomics | Year: 2012

Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one-dimensional (1-D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap-freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlaters. In liver and heart, the protein profiles in 1-D gels were better preserved by snap-freezing, while in gill, the 1-D protein profile was better preserved by immersion in RNAlaters. In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC-MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Xu G.,University of New Orleans | Deng N.,University of New Orleans | Zhao Z.,University of New Orleans | Judeh T.,University of New Orleans | And 4 more authors.
Source Code for Biology and Medicine | Year: 2011

Background: Next Generation Sequencing (NGS) technology generates tens of millions of short reads for each DNA/RNA sample. A key step in NGS data analysis is the short read alignment of the generated sequences to a reference genome. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing this information.Results: We have developed a Graphical User Interface (GUI) software tool named sammate. sammate allows biomedical researchers to quickly process SAM/BAM files and is compatible with both single-end and paired-end sequencing technologies. Sammate also automates some standard procedures in DNA-seq and RNA-seq data analysis. Using either standard or customized annotation files, sammate allows users to accurately calculate the short read coverage of genomic intervals. In particular, for RNA-seq data sammate can accurately calculate the gene expression abundance scores for customized genomic intervals using short reads originating from both exons and exon-exon junctions. Furthermore, sammate can quickly calculate a whole-genome signal map at base-wise resolution allowing researchers to solve an array of bioinformatics problems. Finally, sammate can export both a wiggle file for alignment visualization in the UCSC genome browser and an alignment statistics report. The biological impact of these features is demonstrated via several case studies that predict miRNA targets using short read alignment information files.Conclusions: With just a few mouse clicks, sammate will provide biomedical researchers easy access to important alignment information stored in SAM/BAM files. Our software is constantly updated and will greatly facilitate the downstream analysis of NGS data. Both the source code and the GUI executable are freely available under the GNU General Public License at http://sammate.sourceforge.net. © 2011 Xu et al; licensee BioMed Central Ltd. Source

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