Hox genes from nematodes and RNAi in Brugia malayi

Hox genes are important: playing a central role in the anteroposterior patterning of bodyplans, showing conservation of relative expression pattern and chromosomal order of paralogous groups between phyla, providing the framework for a molecular map of animal body plan evolution and gross morphology. One organism for which this paradigm appears to be inaccurate is in the nematode C. elegans, which has a depauperate Hox cluster compared to other protostomes. The data presented here reveal that Hox genes within the phylum Nematoda are undergoing rapid and dynamic evolution. Hox genes orthologous to those in other protostome phyla have been identified from species representative of a cross-section of the whole of Nematoda, which are definitively absent from C. elegans. This demonstrates loss of multiple Hox genes within the nematode lineage and challenges previously defined Hox signatures for the two major protostome lineages. In addition we observe alternate cis-splicing of the same N-terminal exon to two different C-terminal homeodomains from different Hox orthology groups within the parasitic nematode B. malayi. These findings demonstrate Hox gene loss as an alternative mechanism for the evolution of body plans to gene duplication and changes in coding and regulatory sequences, within the Nematoda. Furthermore the conservation of the simple nematode body plan has not been accompanied by coordinate Hox gene loss through the whole phylum.One problem when working with parasitic species is the lack of molecular genetic techniques available to study gene function directly. A method for studying gene function using the conserved mechanism of RNAi is described and evaluated. The technique successfully results in loss of specific mRNA transcripts from the target organism B. malayi, and allows the observation of resulting phenotypes in culture. This new approach offers the possibility of investigating the roles of developmental genes such as the Hox gene family, or screening for potential novel drug target candidates and should also be applicable to other parasitic helminths

Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

Currently, little is known about the evolution of epigenetic regulation in animal stem cells. Here we demonstrate, using the planarian stem cell system to investigate the role of the COMPASS family of MLL3/4 histone methyltransferases that their function as tumor suppressors in mammalian stem cells is conserved over a long evolutionary distance. To investigate the potential conservation of a genome-wide epigenetic regulatory program in animal stem cells, we assess the effects of Mll3/4 loss of function by performing RNA-seq and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the formation of outgrowths. We find many oncogenes and tumor suppressors among the affected genes that are likely candidates for mediating MLL3/4 tumor suppression function. Our work demonstrates conservation of an important epigenetic regulatory program in animals and highlights the utility of the planarian model system for studying epigenetic regulation

Gtdap-1 and the role of autophagy during planarian regeneration and starvation

Planarians have been established as an ideal model organism for stem cell research and regeneration. Planarian regeneration and homeostasis require an exquisite balancing act between cell death and cell proliferation as new tissues are made (epimorphosis) and existing tissues remodeled (morphallaxis). Some of the genes and mechanisms that control cell proliferation and pattern formation are known. However, studies about cell death during remodeling are few and far between. We have studied the gene Gtdap-1, the planarian ortholog of human death-associated protein-1 or DAP-1. DAP-1 together with DAP-kinase has been identified as a positive mediator of programmed cell death induced by gamma-interferon in HeLa cells. We have found that the gene functions at the interface between autophagy and cell death in the remodeling of the organism that occurs during regeneration and starvation in sexual and asexual races of planarians. Our data suggest that autophagy of existing cells may be essential to fuel the continued proliferation and differentiation of stem cells by providing the necessary energy and building blocks to neoblasts

A Qualitative Investigation into the Lived Experiences of Critical Care Nurses Working on Night Shifts

Background and objectives: Shift work is used in the hospitals and residential treatment centres to provide patients with continuous health care. During their night shift, nurses are often faced with different health problems either due to the stressful work environment or because of the sophisticated instruments to be utilized in intensive care units. The present study was aimed at investigating the lived experience of nurses working on the night shifts at Rizgary Teaching Hospital located in Erbil, Iraqi Kurdistan Region. Methods: A qualitative phenomenological study was conducted to describe the lived experiences of nurses working on night shifts. In doing so and in order to collect the required data, open-ended semi-structured in-depth interviews were carried out to explore thoughts, feelings, and lived experiences of 15 nurses who were working on the night shifts in the Intensive Care Unit (ICU) at Rizgary Teaching Hospital in Erbil. The collected data were then analyzed by Van Manen’s (1990) method. Results: Study participants were between 26 and 38 years old. The majority were male (60%) and 40% were female. Regarding marital status, 10 participants were married and 5 were single. Most of the nurses had Bachelor degrees (86.6%). The respondents work experience was between 3 and 8 years. Almost all of the participating nurses had similar lived experiences during their night shifts. The nurses' lived experiences gave way to the emergence of four themes namely workload, psychosocial issues, sleep disturbance and physiological issues. Conclusion: The present study showed that nurses working on night shifts faced many problems and issues such as poor sleep quality sleep, working for a long time, fatigue and anxiety, back pain, mood disturbance and lack of concentration

Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms

In most sexually reproducing animals, replication and maintenance of telomeres occurs in the germ line and during early development in embryogenesis through the use of telomerase. Somatic cells generally do not maintain telomere sequences, and these cells become senescent in adults as telomeres shorten to a critical length. Some animals reproduce clonally and must therefore require adult somatic mechanisms for maintaining their chromosome ends. Here we study the telomere biology of planarian flatworms with apparently limitless regenerative capacity fueled by a population of highly proliferative adult stem cells. We show that somatic telomere maintenance is different in asexual and sexual animals. Asexual animals maintain telomere length somatically during reproduction by fission or when regeneration is induced by amputation, whereas sexual animals only achieve telomere elongation through sexual reproduction. We demonstrate that this difference is reflected in the expression and alternate splicing of the protein subunit of the telomerase enzyme. Asexual adult planarian stem cells appear to maintain telomere length over evolutionary timescales without passage through a germ-line stage. The adaptations we observe demonstrate indefinite somatic telomerase activity in proliferating stem cells during regeneration or reproduction by fission, and establish planarians as a pertinent model for studying telomere structure, function, and maintenance

Organ specific gene expression in the regenerating tail of Macrostomum lignano

Temporal and spatial characterization of gene expression is a prerequisite for the understanding of cell-, tissue-, and organ-differentiation. In a multifaceted approach to investigate gene expression in the tail plate of the free-living marine flatworm Macrostomum lignano, we performed a posterior-region-specific in situ hybridization screen, RNA sequencing (RNA-seq) of regenerating animals, and functional analyses of selected tail-specific genes. The in situ screen revealed transcripts expressed in the antrum, cement glands, adhesive organs, prostate glands, rhabdite glands, and other tissues. Next we used RNA-seq to characterize temporal expression in the regenerating tail plate revealing a time restricted onset of both adhesive organs and copulatory apparatus regeneration. In addition, we identified three novel previously unannotated genes solely expressed in the regenerating stylet. RNA interference showed that these genes are required for the formation of not only the stylet but the whole male copulatory apparatus. RNAi treated animals lacked the stylet, vesicula granulorum, seminal vesicle, false seminal vesicle, and prostate glands, while the other tissues of the tail plate, such as adhesive organs regenerated normally. In summary, our findings provide a large resource of expression data during homeostasis and regeneration of the morphologically complex tail regeneration and pave the way for a better understanding of organogenesis in M. lignano

A Dual Platform Approach to Transcript Discovery for the Planarian Schmidtea Mediterranea to Establish RNAseq for Stem Cell and Regeneration Biology

The use of planarians as a model system is expanding and the mechanisms that control planarian regeneration are being elucidated. The planarian Schmidtea mediterranea in particular has become a species of choice. Currently the planarian research community has access to this whole genome sequencing project and over 70,000 expressed sequence tags. However, the establishment of massively parallel sequencing technologies has provided the opportunity to define genetic content, and in particular transcriptomes, in unprecedented detail. Here we apply this approach to the planarian model system. We have sequenced, mapped and assembled 581,365 long and 507,719,814 short reads from RNA of intact and mixed stages of the first 7 days of planarian regeneration. We used an iterative mapping approach to identify and define de novo splice sites with short reads and increase confidence in our transcript predictions. We more than double the number of transcripts currently defined by publicly available ESTs, resulting in a collection of 25,053 transcripts described by combining platforms. We also demonstrate the utility of this collection for an RNAseq approach to identify potential transcripts that are enriched in neoblast stem cells and their progeny by comparing transcriptome wide expression levels between irradiated and intact planarians. Our experiments have defined an extensive planarian transcriptome that can be used as a template for RNAseq and can also help to annotate the S. mediterranea genome. We anticipate that suites of other 'omic approaches will also be facilitated by building on this comprehensive data set including RNAseq across many planarian regenerative stages, scenarios, tissues and phenotypes generated by RNAi

Downregulation of mTOR Signaling Increases Stem Cell Population Telomere Length during Starvation of Immortal Planarians

Reduction of caloric intake delays and prevents age-associated diseases and extends the life span in many organisms. It may be that these benefits are due to positive effects of caloric restriction on stem cell function. We use the planarian model Schmidtea mediterranea, an immortal animal that adapts to long periods of starvation by shrinking in size, to investigate the effects of starvation on telomere length. We show that the longest telomeres are a general signature of planarian adult stem cells. We also observe that starvation leads to an enrichment of stem cells with the longest telomeres and that this enrichment is dependent on mTOR signaling. We propose that one important effect of starvation for the rejuvenation of the adult stem cell pool is through increasing the median telomere length in somatic stem cells. Such a mechanism has broad implications for how dietary effects on aging are mediated at the whole-organism level.C.G.-E. was funded by a Contrato de Investigadores Miguel Servet (CP12/03214) and by the FLI. The FLI is a member of the Leibniz Association and is financially supported by the Federal Government of Germany and the State of Thuringia. O.G.-G. was funded by an LGSA scholarship. R.P. and B.F.-V. were funded by a grant (PI17-01401) from Fondo de Investigaciones Sanitarias (Instituto de Salud Carlos III, Spain) and FEDER funds. I.F. was funded by grants from Ministerio de Ciencia, Innovación y Universidades (SAF2016-80406-R), Comunidad de Madrid (S2017/BMD-3875), and the Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares (RD12/0042/0045). The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). A.A.A. was funded by grants from the BBSRC (BB/K007564/1) and MRC (MR/M000133/1), and S.S. by a University of Oxford Clarendon Fund Scholarship.S

No evidence for extensive horizontal gene transfer in the genome of the tardigrade <i>Hypsibius dujardini</i>

No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini These files accompany the peer-reviewed version of http://dx.doi.org/10.1101/033464 A previous dataset https://zenodo.org/record/45162 accompanied the version of this manuscript at BioRxiv - biorxiv.org/content/early/2015/12/13/033464 This dataset includes all files from https://zenodo.org/record/45162 plus all the Supplemental files, and one additional file HGT_phylogenetic_files.tgz. All files are described in Hypsibius_dujardini_files_README.md Abstract Tardigrades are meiofaunal ecdysozoans that are key to understanding the origins of Arthropoda. Many species of Tardigrada can survive extreme conditions through cryptobiosis. In a recent paper (Boothby TC et al (2015) Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade. Proc Natl Acad Sci USA 112:15976-15981) the authors concluded that the tardigrade Hypsibius dujardini had an unprecedented proportion (17%) of genes originating through functional horizontal gene transfer (fHGT), and speculated that fHGT was likely formative in the evolution of cryptobiosis. We independently sequenced the genome of H. dujardini. As expected from whole-organism DNA sampling, our raw data contained reads from non-target genomes. Filtering using metagenomics approaches generated a draft H. dujardini genome assembly of 135 Mb with superior assembly metrics to the previously published assembly. Additional microbial contamination likely remains. We found no support for extensive fHGT. Among 23,021 gene predictions we identified 0.2% strong candidates for fHGT from bacteria, and 0.2% strong candidates for fHGT from non-metazoan eukaryotes. Cross-comparison of assemblies showed that the overwhelming majority of HGT candidates in the Boothby et al. genome derived from contaminants. We conclude that fHGT into H. dujardini accounts for at most 1-2% of genes and that the proposal that one sixth of tardigrade genes originate from functional HGT events is an artefact of undetected contamination