The analysis of pendolino (peo) mutants reveals differences in the fusigenic potential among Drosophila telomeres

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peodependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peodepleted cells results in specific fusigenic lesions concentrated in heterochromatinassociated telomeres. Alternatively it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping

AKTIP/Ft1, a new shelterin-interacting factor required for telomere maintenance

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (humanAKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factoridentified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere disfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication

Spatially resolved transcriptomics reveals innervation-responsive functional clusters in skeletal muscle

Striated muscle is a highly organized structure composed of well-defined anatomical domains with integrated but distinct assignments. So far, the lack of a direct correlation between tissue architecture and gene expression has limited our understanding of how each unit responds to physio-pathologic contexts. Here, we show how the combined use of spatially resolved transcriptomics and immunofluorescence can bridge this gap by enabling the unbiased identification of such domains and the characterization of their response to external perturbations. Using a spatiotemporal analysis, we follow changes in the transcriptome of specific domains in muscle in a model of denervation. Furthermore, our approach enables us to identify the spatial distribution and nerve dependence of atrophic signaling pathway and polyamine metabolism to glycolytic fibers. Indeed, we demonstrate that perturbations of polyamine pathway can affect muscle function. Our dataset serves as a resource for future studies of the mechanisms underlying skeletal muscle homeostasis and innervation

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response

Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin

Prevalence, Related Factors, and Levels of Burnout Syndrome Among Nurses Working in Gynecology and Obstetrics Services: A Systematic Review and Meta-Analysis

Background: Although burnout levels and the corresponding risk factors have been studied in many nursing services, to date no meta-analytical studies have been undertaken of obstetrics and gynecology units to examine the heterogeneity of burnout in this environment and the variables associated with it. In the present paper, we aim to determine the prevalence, levels, and related factors of burnout syndrome among nurses working in gynecology and obstetrics services. Methods: A systematic review and meta-analysis of the literature were carried out using the following sources: CINAHL (Cumulative Index of Nursing and Allied Health Literature), LILACS (Latin American and Caribbean Health Sciences Literature), Medline, ProQuest (Proquest Health and Medical Complete), SciELO (Scientific Electronic Library Online), and Scopus. Results: Fourteen relevant studies were identified, including, for this meta-analysis, n = 464 nurses. The following prevalence values were obtained: emotional exhaustion 29% (95% CI: 11–52%), depersonalization 19% (95% CI: 6–38%), and low personal accomplishment 44% (95% CI: 18–71%). The burnout variables considered were sociodemographic (age, marital status, number of children, gender), work-related (duration of the workday, nurse-patient ratio, experience or number of miscarriages/abortions), and psychological (anxiety, stress, and verbal violence). Conclusion: Nurses working in obstetrics and gynecology units present high levels of burnout syndrome. In over 33% of the study sample, at least two of the burnout dimensions considered are apparent.This research was funded by the Excellence Research Project (P11HUM-7771) provided by the Andalusian Government (Spain)

Identification of Drosophila Mitotic Genes by Combining Co-Expression Analysis and RNA Interference

RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression–based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression–based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins

An essential function of AP-1 heterodimers in Drosophila.

Fos and Jun proteins homo- or heterodimerize to form functional AP-1 transcription factors. Drosophila mutants lacking either Jun or Fos display indistinguishable dorsal open phenotypes, indicating an essential function of both Jun and Fos for embryonic dorsal closure. Here we present experiments to determine the basis for this dual requirement. By combining mutant alleles and transgenes expressing Fos and Jun variants with altered dimerization preferences, fly lines were generated in which only specifically defined dimer variants can form. Phenotypic analysis of these mutants reveals that homodimers of Fos or of Jun cannot replace the function of the heterodimeric complex. This defect is not explained by the lower stability of homodimers as compared to heterodimers, because 'pseudo-homodimers' which are as stable as native Jun-Fos heterodimers cannot substitute for their function. We conclude that Jun and Fos play complementary roles that are both required for signal transduction and gene activation during dorsal closure

Telomere capping and cellular checkpoints: clues from fruit flies

In most organisms, telomeres consist of repetitive G-rich sequences that are elongated by a specific reverse transcriptase, telomerase. A large number of proteins are recruited by these terminal repeats, forming specialized structures that regulate telomerase activity and protect telomeres from degradation and recombination. Drosophila lacks telomerase and telomere length is maintained by transposition of three specialized retrotransposons. In addition, unlike yeast and mammals, Drosophila telomeres are epigenetically determined, sequence-independent structures. However, several proteins required for Drosophila telomere behavior are evolutionarily conserved. These include the Mre11-Rad50-Nbs (MRN) complex and the Ataxia Telangiectasia Mutated (ATM) kinase, which are required to prevent telomeric fusions. In addition, recent studies have provided evidence that Drosophila uncapped telomeres elicit a DNA damage response (DDR) just as dysfunctional yeast and human telomeres. Uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. Telomere-induced DDR and SAC both require the wild type function of the MRN complex. In addition, while DDR is mediated by ATR kinase, SAC activation requires both the ATM and ATR activities. These results indicate that the DNA repair systems play multiple roles at Drosophila telomeres, highlighting the importance of this model organism for investigations on the relationships between DNA repair and telomere maintenance

The Drosophila Nbs Protein Functions in Multiple Pathways for the Maintenance of Genome Stability

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations

The mechanism of telomere protection: a comparison between Drosophila and humans

Drosophila telomeres are maintained by transposition of specialized retrotransposons rather than by telomerase activity, and their stability is independent of the sequence of DNA termini. Recent studies have identified several proteins that protect Drosophila telomeres from fusion events. These proteins include the telomere capping factors HP1/ORC-associated protein (HOAP) and heterochromatin protein 1 (HP1), the Rad50 and Mre11 DNA repair proteins that are required for HOAP and HP1 localization at telomeres, and the ATM kinase. Another telomere- protecting factor identified in Drosophila is UbcD1, a polypeptide highly homologous to class I ubiquitin-conjugating E2 enzymes. In addition, it has been shown that HP1 and both components of the Drosophila Ku70/80 heterodimer act as negative regulators of telomere length. Except for HOAP, all these proteins are conserved in humans and are associated with human telomeres. Collectively, these results indicate that Drosophila is an excellent model system for the analysis of the mechanisms of telomere maintenance. In past and current studies, 15 Drosophila genes have been identified that prevent telomeric fusion, and it has been estimated that the Drosophila genome contains at least 40 genes required for telomere protection. We believe that the molecular characterization of these genes will lead to identification of many novel human genes with roles in telomere maintenance