Synthesizing and Salvaging NAD+: Lessons Learned from Chlamydomonas reinhardtii

The essential coenzyme nicotinamide adenine dinucleotide (NAD+) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD+. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD+ synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1) or plasmids with cloned genes (nic1-1 and nic15-1) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD+. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD+ metabolism and cell longevity

Improving Gene-finding in Chlamydomonas reinhardtii:GreenGenie2

<p>Abstract</p> <p>Background</p> <p>The availability of whole-genome sequences allows for the identification of the entire set of protein coding genes as well as their regulatory regions. This can be accomplished using multiple complementary methods that include ESTs, homology searches and <it>ab initio </it>gene predictions. Previously, the Genie gene-finding algorithm was trained on a small set of <it>Chlamydomonas </it>genes and shown to improve the accuracy of gene prediction in this species compared to other available programs. To improve <it>ab initio </it>gene finding in <it>Chlamydomonas</it>, we assemble a new training set consisting of over 2,300 cDNAs by assembling over 167,000 <it>Chlamydomonas </it>EST entries in GenBank using the EST assembly tool PASA.</p> <p>Results</p> <p>The prediction accuracy of our cDNA-trained gene-finder, GreenGenie2, attains 83% sensitivity and 83% specificity for exons on short-sequence predictions. We predict about 12,000 genes in the version <it>v3 Chlamydomonas </it>genome assembly, most of which (78%) are either identical to or significantly overlap the published catalog of <it>Chlamydomonas </it>genes <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. 22% of the published catalog is absent from the GreenGenie2 predictions; there is also a fraction (23%) of GreenGenie2 predictions that are absent from the published gene catalog. Randomly chosen gene models were tested by RT-PCR and most support the GreenGenie2 predictions.</p> <p>Conclusion</p> <p>These data suggest that training with EST assemblies is highly effective and that GreenGenie2 is a valuable, complementary tool for predicting genes in <it>Chlamydomonas reinhardtii</it>.</p

Short-Term Serial Sampling of Natriuretic Peptides in Patients Presenting With Chest Pain

ObjectivesThe purpose of this study was to characterize the diagnostic and prognostic utility of short-term dynamic changes in natriuretic peptides in patients presenting with chest pain.BackgroundAlthough single levels of natriuretic peptides in patients admitted for acute coronary syndromes (ACS) have important prognostic value, it is unclear whether serial sampling of natriuretic peptides might have both diagnostic and prognostic value in the setting of chest pain.MethodsWe followed 276 patients for 90 days who presented to the emergency department with chest pain. We sampled brain natriuretic peptide (BNP) and amino-terminal (NT)-proBNP up to 5 times within 24 h of presentation and again at discharge. Follow-up data was collected at 30 and 90 days after admission. Adverse events included emergency department visits for chest pain, cardiac readmission, and death. We assessed the prognostic and diagnostic value of baseline natriuretic peptide measurements with receiver-operating characteristic analyses.ResultsNatriuretic peptides were diagnostic for congestive heart failure (CHF) and new-onset CHF but less so for ACS. The prognostic utility of serial sampling was evaluated through testing the statistical contribution of each future time point (as well as variability over time) over and above the baseline values in logistic regression models.ConclusionsBaseline elevated BNP and NT-proBNP concentrations were predictive of adverse events at 30 and 90 days. Serial sampling did not improve the prognostic value of BNP or NT-proBNP

Identification of cilia genes that affect cell-cycle progression using whole-genome transcriptome analysis in Chlamydomonas reinhardtti

Cilia are microtubule based organelles that project from cells. Cilia are found on almost every cell type of the human body and numerous diseases, collectively termed ciliopathies, are associated with defects in cilia, including respiratory infections, male infertility, situs inversus, polycystic kidney disease, retinal degeneration, and Bardet-Biedl Syndrome. Here we show that Illumina-based whole-genome transcriptome analysis in the biflagellate green alga Chlamydomonas reinhardtii identifies 1850 genes up-regulated during ciliogenesis, 4392 genes down-regulated, and 4548 genes with no change in expression during ciliogenesis. We examined four genes up-regulated and not previously known to be involved with cilia (ZMYND10, NXN, GLOD4, SPATA4) by knockdown of the human orthologs in human retinal pigment epithelial cells (hTERT-RPE1) cells to ask whether they are involved in cilia-related processes that include cilia assembly, cilia length control, basal body/centriole numbers, and the distance between basal bodies/centrioles. All of the genes have cilia-related phenotypes and, surprisingly, our data show that knockdown of GLOD4 and SPATA4 also affects the cell cycle. These results demonstrate that whole-genome transcriptome analysis during ciliogenesis is a powerful tool to gain insight into the molecular mechanism by which centrosomes and cilia are assembled

Whole-Genome Sequencing to Identify Mutants and Polymorphisms in Chlamydomonas reinhardtii

Whole-genome sequencing (WGS) provides a new platform for the identification of mutations that produce a mutant phenotype. We used Illumina sequencing to identify the mutational profile of three Chlamydomonas reinhardtii mutant strains. The three strains have more than 38,000 changes from the reference genome. NG6 is aflagellate and maps to 269 kb with only one nonsynonymous change; the V12E mutation falls in the FLA8 gene. Evidence that NG6 is a fla8 allele comes from swimming revertants that are either true or pseudorevertants. NG30 is aflagellate and maps to 458 kb that has six nonsynonomous changes. Evidence that NG30 has a causative nonsense allele in IFT80 comes from rescue of the nonswimming phenotype with a fragment bearing only this gene. This gene has been implicated in Jeune asphyxiating thoracic dystrophy. Electron microscopy of ift80-1 (NG30) shows a novel basal body phenotype. A bar or cap is observed over the distal end of the transition zone, which may be an intermediate in preparing the basal body for flagellar assembly. In the acetate-requiring mutant ac17, we failed to find a nonsynonymous change in the 676 kb mapped region, which is incompletely assembled. In these strains, 43% of the changes occur on two of the 17 chromosomes. The excess on chromosome 6 surrounds the mating-type locus, which has numerous rearrangements and suppressed recombination, and the changes extend beyond the mating-type locus. Unexpectedly, chromosome 16 shows an unexplained excess of single nucleotide polymorphisms and indels. Overall, WGS in combination with limited mapping allows fast and accurate identification of point mutations in Chlamydomonas

Global analysis of patterns of gene expression during Drosophila embryogenesis

Embryonic expression patterns for 6,003 (44%) of the 13,659 protein-coding genes identified in the Drosophila melanogaster genome were documented, of which 40% show tissue-restricted expression

Proteomic analyses of primary human villous trophoblasts exposed to flame retardant BDE-47 using SWATH-MS

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants and recognized developmental toxicants that are detectable in placental tissues. Higher levels of in utero PBDE exposure have been associated with an increased risk of adverse birth outcomes. During pregnancy, cytotrophoblasts (CTBs) from the placenta play critical roles in the formation of the maternal-fetal interface via uterine invasion and vascular remodeling. The differentiation of these cells towards an invasive phenotype is crucial for proper placental development. We previously have shown that BDE-47 can impact CTB viability and hinder the ability of these cells to migrate and invade. To expand on potential toxicological mechanisms, we utilized quantitative proteomic approaches to identify changes in the global proteome of mid-gestation primary human CTBs after exposure to BDE-47. Using sequential window acquisition of all theoretical fragment-ion spectra (SWATH), we identified 3024 proteins in our CTB model of differentiation/invasion. Over 200 proteins were impacted as a function of BDE-47 exposure (1&nbsp;μM and 5&nbsp;μM) across the treatment period (15, 24, and 39&nbsp;h). The differentially expressed molecules displayed time- and concentration-dependent changes in expression and were enriched in pathways associated with aggregatory and adhesive processes. Network analysis identified CYFIP1, a molecule previously unexplored in a placental context, to be dysregulated at BDE-47 concentrations previously seen to impact CTB migration/invasion. Our SWATH-MS dataset thus demonstrates BDE-47 impacts the global proteome of differentiating CTBs and serves as a valuable resource for further understanding of the relationship between environmental chemical exposures and placental development and function. AVAILABILITY OF DATA AND MATERIAL: Raw chromatograms are deposited on the MassIVE proteomic database (https://massive.ucsd.edu) under accession number MSV000087870. Normalized relative abundances are also available as Table S1

Effectiveness of a Faculty Development Program in Fostering Interprofessional Education Competencies

AbstractBackground: To determine the effectiveness of a faculty development program offered to clinical faculty in fostering interprofessional education competencies.Methods and Findings: A pre-post randomized control group design was used in which only one of two cohorts of clinical faculty received an interprofessional educational intervention. Both cohorts then facilitated case-based interprofessional education sessions for student learners. A variety of outcome measures were used to assess differences between groups in terms of knowledge, skills, and attitudes related to interprofessional education and practice. No significant differences were noted between the control and intervention groups.Conclusions: The use of a pre-post randomized control group design to measure effectiveness of an educational intervention should be considered to demonstrate the impact of educational interventions

Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma.

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development