Mutations in tyrosyl-DNA phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during Caenorhabditis elegans spermatogenesis.

Meiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5'-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.

TOP-2 is differentially required for the proper maintenance of the cohesin subunit REC-8 on meiotic chromosomes in Caenorhabditis elegans spermatogenesis and oogenesis.

During meiotic prophase I, accurate segregation of homologous chromosomes requires the establishment of chromosomes with a meiosis-specific architecture. The sister chromatid cohesin complex and the enzyme Topoisomerase II (TOP-2) are important components of meiotic chromosome architecture, but the relationship of these proteins in the context of meiotic chromosome segregation is poorly defined. Here, we analyzed the role of TOP-2 in the timely release of the sister chromatid cohesin subunit REC-8 during spermatogenesis and oogenesis of Caenorhabditis elegans. We show that there is a different requirement for TOP-2 in meiosis of spermatogenesis and oogenesis. The loss-of-function mutation top-2(it7) results in premature REC-8 removal in spermatogenesis, but not oogenesis. This correlates with a failure to maintain the HORMA-domain proteins HTP-1 and HTP-2 (HTP-1/2) on chromosome axes at diakinesis and mislocalization of the downstream components that control REC-8 release including Aurora B kinase. In oogenesis, top-2(it7) causes a delay in the localization of Aurora B to oocyte chromosomes but can be rescued through premature activation of the maturation promoting factor via knockdown of the inhibitor kinase WEE-1.3. The delay in Aurora B localization is associated with an increase in the length of diakinesis bivalents and wee-1.3 RNAi mediated rescue of Aurora B localization in top-2(it7) is associated with a decrease in diakinesis bivalent length. Our results imply that the sex-specific effects of TOP-2 on REC-8 release are due to differences in the temporal regulation of meiosis and chromosome structure in late prophase I in spermatogenesis and oogenesis.

Revisiting the Innovation Strategy performance measurement process: insights from practice.

Performance measurement (PM) aims to ensure transparency and effectiveness in public spending. More specifically, it provides a foundation that allows funders to determine whether an intervention has achieved its stated objective, thus improving understanding of what interventions should be funded in the future. Ideally, performance measurement should help us understand "what works, for whom, and in what context." Unfortunately, more often than not, performance reports are limited to a collection of indicators that make it difficult to answer this question. Based on our work with the Public Health Agency of Canada Innovation Strategy (PHAC-IS), we offer several recommendations to address this issue and support the performance measurement process, especially for complex interventions. We discuss the importance of contextualizing results to better understand impact and associating these results to a well-defined intervention. We also suggest using a validated tool to improve data collection and analysis and critically appraise the methods used to collect impact information. The integration of these key considerations will save time in data analysis and ensure funded recipients are not overburdened by the data collection process. Although this commentary is presented within the context of a complex multi-year population health funding program, we believe our approach can be applied to any performance management process and ultimately improve decisions such as whether an intervention should be continued, scaled up, or adapted to different contexts.

RéSUMé: La mesure du rendement vise à garantir la transparence et l'efficacité des dépenses publiques. Plus précisément, elle fournit une base qui permet aux bailleurs de fonds de déterminer si une intervention a atteint son objectif énoncé, améliorant ainsi la compréhension des interventions qui devraient être financées à l'avenir. Idéalement, la mesure du rendement devrait nous aider à comprendre « ce qui fonctionne, pour qui et dans quel contexte ¼. Malheureusement, la plupart du temps, les rapports sur le rendement se limitent à un ensemble d'indicateurs qui font en sorte qu'il est difficile de répondre à cette question. Sur la base de notre travail avec la Stratégie d'innovation de l'Agence de la santé publique du Canada (SI de l'ASPC), nous proposons plusieurs recommandations pour traiter cette question et soutenir le processus de mesure du rendement, en particulier pour les interventions complexes. Nous discutons de l'importance de contextualiser les résultats pour mieux comprendre les répercussions et d'associer ces résultats à une intervention bien définie. De plus, nous suggérons également d'utiliser un outil validé pour améliorer la collecte et l'analyse des données, et évaluer de manière critique les méthodes utilisées pour recueillir les renseignements sur les répercussions. L'intégration de ces considérations clés permettra de gagner du temps dans l'analyse des données et garantira que les bénéficiaires financés ne seront pas surchargés par le processus de collecte de données. Bien que le présent commentaire soit présenté dans le contexte d'un programme pluriannuel complexe en matière de financement de la santé de la population, nous pensons que notre approche peut être appliquée à tout processus de gestion du rendement et, en fin de compte, améliorer les décisions visant à déterminer si une intervention devrait être poursuivie, étendue à plus grande échelle ou adaptée à différents contextes.

Endogenous localization of TOP-2 in C. elegans using a C-terminal GFP-tag.

To investigate the dynamic localization of Topoisomerase II in live C. elegans we have generated a C-terminally GFP-tagged version of TOP-2 at the endogenous locus. We found that TOP-2::GFP localizes in a similar pattern to the previously published TOP-2::3XFLAG strain and does not disrupt the meiotic chromosome segregation functions of this enzyme.

Identification of Suppressors of top-2 Embryonic Lethality in Caenorhabditis elegans.

Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.

Changing the conversation about hunger: the process of developing Freshplace.

BACKGROUND: Despite public and private food assistance programs, food insecurity and hunger are persistent public health problems. Freshplace is an innovative food pantry collaborative whose goal is to build long-term food security and self-sufficiency among residents of the North End of Hartford, Connecticut. Freshplace was founded by Foodshare (FS), the Chrysalis Center (CC), Inc., and the Junior League of Hartford (JLH), Inc., who then partnered with the University of Connecticut to design and evaluate the program. OBJECTIVES: This article describes the community-based participatory research process involved with developing and evaluating Freshplace. METHODS: We are conducting a randomized, controlled study to compare 100 Freshplace members with 100 people who receive food from traditional food pantries. Main outcome measures include food security, self-sufficiency, and diet quality. Change scores are compared from baseline to 3 months using independent t tests. RESULTS: Freshplace opened in July 2010. We have recruited 233 people to participate in the study. Over 3 months, Freshplace members had larger change scores than the comparison group in food security scores (1.6 vs. 0.7 points; p < .01), and fruit and vegetable intake (1.9 vs. -1.4 points; p < .01). CONCLUSIONS: Freshplace is a successful, community- university partnership bringing together three community agencies and a state university. This is the first food pantry intervention to be evaluated, and preliminary results are promising.

Methods for predicting sexual maturity in male cynomolgus macaques on the basis of age, body weight, and histologic evaluation of the testes.

In pharmaceutical drug safety testing, sexual maturity is an important experimental parameter. Histologic immaturity of the tissues of the reproductive system can interfere with the interpretation of compound-related effects on the reproductive organs. In female cynomolgus macaques, determination of sexual maturity is simplified by the presence of a menstrual cycle. For male cynomolgus macaques, predicting maturity is much more difficult. In this study, we evaluated methods that would reliably predict sexual maturity in male cynomolgus macaques. The results of histologic examination of testes of control male cynomolgus macaques used for drug safety studies were examined retrospectively for evidence of sexual maturity. These data were compared with age and body weight determinations to establish statistical models for determining the probability that a male cynomolgus macaque is sexually mature. This model presents a simple prospective method of predicting sexual maturity in male cynomolgus macaques.