Correlates of Complete Childhood Vaccination in East African Countries.

Despite the benefits of childhood vaccinations, vaccination rates in low-income countries (LICs) vary widely. Increasing coverage of vaccines to 90% in the poorest countries over the next 10 years has been estimated to prevent 426 million cases of illness and avert nearly 6.4 million childhood deaths worldwide. Consequently, we sought to provide a comprehensive examination of contemporary vaccination patterns in East Africa and to identify common and country-specific barriers to complete childhood vaccination. Using data from the Demographic and Health Surveys (DHS) for Burundi, Ethiopia, Kenya, Rwanda, Tanzania, and Uganda, we looked at the prevalence of complete vaccination for polio, measles, Bacillus Calmette-Guérin (BCG) and DTwPHibHep (DTP) as recommended by the WHO among children ages 12 to 23 months. We conducted multivariable logistic regression within each country to estimate associations between complete vaccination status and health care access and sociodemographic variables using backwards stepwise regression. Vaccination varied significantly by country. In all countries, the majority of children received at least one dose of a WHO recommended vaccine; however, in Ethiopia, Tanzania, and Uganda less than 50% of children received a complete schedule of recommended vaccines. Being delivered in a public or private institution compared with being delivered at home was associated with increased odds of complete vaccination status. Sociodemographic covariates were not consistently associated with complete vaccination status across countries. Although no consistent set of predictors accounted for complete vaccination status, we observed differences based on region and the location of delivery. These differences point to the need to examine the historical, political, and economic context of each country in order to maximize vaccination coverage. Vaccination against these childhood diseases is a critical step towards reaching the Millennium Development Goal of reducing under-five mortality by two-thirds by 2015 and thus should be a global priority

Multi-state gene cluster switches determine the adaptive mitochondrial and metabolic landscape of breast cancer

Adaptive metabolic switches are proposed to underlie conversions between cellular states during normal development as well as in cancer evolution. Metabolic adaptations represent important therapeutic targets in tumors, highlighting the need to characterize the full spectrum, characteristics, and regulation of the metabolic switches. To investigate the hypothesis that metabolic switches associated with specific metabolic states can be recognized by locating large alternating gene expression patterns, we developed a method to identify interspersed gene sets by massive correlated biclustering (MCbiclust) and to predict their metabolic wiring. Testing the method on breast cancer transcriptome datasets revealed a series of gene sets with switch-like behavior that could be used to predict mitochondrial content, metabolic activity, and central carbon flux in tumors. The predictions were experimentally validated by bioenergetic profiling and metabolic flux analysis of 13C-labelled substrates. The metabolic switch positions also distinguished between cellular states, correlating with tumor pathology, prognosis, and chemosensitivity. The method is applicable to any large and heterogeneous transcriptome dataset to discover metabolic and associated pathophysiological states

The role of homologous recombination in the maintenance and repair of the mitochondrial genome

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2017.Mitochondria are dynamic and multifunctional organelles. While the mitochondrion is traditionally thought of as the “power house” of the cell, it has many functions beyond this, including roles in the biosynthesis of amino acids and lipids, signalling, and calcium homeostasis. Mitochondria also contain their own genome that must be replicated and repaired faithfully. Mutations and deletions in the mitochondrial genome are associated with a number of human diseases and syndromes. Homologous recombination is an essential process in the nucleus that facilitates DNA replication, repair, and chromosome segregation. The contribution of homologous recombination to mitochondrial DNA (mtDNA) metabolism is less well understood in comparison. Using our direct repeat mediated deletion assay and an optimized mitochondrial induced double-strand break assay, I have characterized the role of Rad51p, Rad52p, Rad59p, Cce1p, and Irc3p in mitochondrial homologous recombination. Based on our findings, we have concluded that both Rad51p and Rad59p are localized to the matrix of the mitochondria, and that Rad51p and Irc3p bind directly to mtDNA. We find that loss of each of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs. Cce1p prevents spontaneous deletions from occurring under certain growth conditions, suggesting that the overall metabolic state of the cell can influence the stability of the mtDNA. Irc3p is critical to the overall stability of the mitochondrial genome. Without Irc3p, the frequency of spontaneous deletions and respiration loss is extremely high, which suggests that Irc3p may play a critical role in several aspects of mitochondrial homologous recombination. Taken together these studies have revealed that homologous recombination proteins function in diverse pathways that all impact mtDNA metabolism

Members of the <i>RAD52</i> Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in <i>Saccharomyces cerevisiae</i>

<div><p>Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-<i>Kpn</i>I) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair <i>in vivo</i> in <i>Saccharomyces cerevisiae</i>. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.</p></div

Average fold change of amount of 3’ ssDNA at <i>Kpn</i>I cleavage site after induction.

<p>^a<i>p</i> values were calculated were calculated by un-paired, two-tailed t-tests.</p><p>Average fold change of amount of 3’ ssDNA at <i>Kpn</i>I cleavage site after induction.</p

Conversion of DSBs to deletion product is impaired in HR mutants.

<p>Experiments were performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005664#pgen.1005664.g004" target="_blank">Fig 4</a>. The ratios of the amount of <i>COX2</i> signal in the cut band at t = 0 and the amount of <i>COX2</i> signal in the deletion product at t = 8 were calculated. The intensity of the <i>COX2</i>-hybridizing bands was measured using Image Lab software (<a href="http://www.biorad.com" target="_blank">www.biorad.com</a>). The proportions of the total <i>COX2</i> signal present in the DSB and deletion products were calculated as a percent of the total <i>COX2</i> signal. Error bars indicate SD. Asterisks indicate significant differences between the mutant and wild-type rates (* = <i>p</i> ≤ 0.05, *** = <i>p</i> ≤ 0.001).</p

Models for the generation of deletions between directly repeated sequences.

<p>Repair of a double-strand break (DSB) located between directly repeated sequences (grey boxes) can result in the deletion of one of the repeats and all of the intervening sequence. 5’ to 3’ resection at the DSB reveals the direct repeats. Rad52p alone or in conjunction with Rad59p, promotes the annealing of the repeats. Template switching can occur if a lesion (yellow star) is encountered during replication. At a stalled fork, the nascent strand may invade at the incorrect repeat, leading to the generation of a deletion. Unequal exchange can occur when HR occurs between misaligned repeats leading to a deletion. Both template switching and unequal exchange can happen intramolecularly or intermolecularly. Arrowheads indicate 3’ end.</p

Inducing a specific mitochondrial DSB.

<p>(A) The mitochondrial DRMD reporter, with restriction recognition sequences indicated. Horizontal line beneath <i>COX2</i> indicates where the <i>COX2</i> probe anneals. (B) Representative Southern blot of <i>Ava</i>II-digested DNA extracted from EAS748 containing pEAS115 (mtLS-<i>Kpn</i>I-intein^dead) or pEAS114 (mtLS-<i>Kpn</i>I-intein^ts). Both strains were grown in SRaffinose-Arg-Ura until OD₆₀₀ = 0.150. Galactose was added to a final concentration of 2%, and the strains were shifted to 20°C to induce the either mtLS-<i>Kpn</i>I-intein^ts or mtLS-<i>Kpn</i>I-intein^dead. Genomic DNA in lanes labeled P are from preinduced cultures. Cultures were incubated for 16–18 hours at 20°C and samples were taken for the time 0 timepoint. The cultures then were shifted to 30°C to allow repair. Lane I contains DNA from the t = 0 timepoint of the mtLS-<i>Kpn</i>I-intein^ts-containing strain digested <i>in vitro</i> with <i>Kpn</i>I and <i>Ava</i>II to demonstrate the migration of DNA with a DSB. The 21S rRNA gene was probed to detect total mtDNA. The 25S rRNA gene was probed to detect total nuclear DNA. (C) Relative mtDNA content was determined by taking the ratio of the 21S rRNA signal and the 25S rRNA signal, then normalizing to the t = 0 sample for each strain. (D) The intensity of the <i>COX2</i>-hybridizing bands were measured using Image Lab software (<a href="http://www.biorad.com" target="_blank">www.biorad.com</a>) The proportions of the total <i>COX2</i> signal present in the DSB and deletion products were calculated as a percent of the total <i>COX2</i> signal.</p