Two waves of de novo methylation during mouse germ cell development

During development, mammalian germ cells reprogram their epigenomes via a genome-wide erasure and de novo rewriting of DNA methylation marks. We know little of how methylation patterns are specifically determined. The piRNA pathway is thought to target the bulk of retrotransposon methylation. Here we show that most retrotransposon sequences are modified by default de novo methylation. However, potentially active retrotransposon copies evade this initial wave, likely mimicking features of protein-coding genes. These elements remain transcriptionally active and become targets of piRNA-mediated methylation. Thus, we posit that these two waves play essential roles in resetting germ cell epigenomes at each generation

Effects of intersensory localization of spheres and prisms as measured in Harris type apparatus

The vision specialist often finds himself asking the question, What are the immediate effects of my lens and/or prism therapy on a given patient? If for example, a change in accommodation or convergence is effected by lenses, what will be the results of this change on the visual performance of the patient as far as his intersensory localizations of objects in space are concerned? Past experience by some traditional practitioners would dictate that positive lenses and prism base-in will tend to force a subject to localize farther out than his habitual localization pattern. Minus lenses·and prism base-out tend to localize closer than he normally would. This effect, they would say, is an illustration of the phenomenon known as SILO. The letters SILO stand for the phenomenon of smaller-in and larger-out. For example, if a subject views an object through minus spherical lenses or base-out prisms, he will experience the object as being smaller and closer whereas if he views the same object through plus spherical lenses or base-in prisms, he will experience the object as being larger and farther away. Our thesis deals only with half of the SILO effect, i.e., the perceived distance. Recently, some developmentalists have postulated that localization may be attributable to postural functions of accommodation. Specifically, since plus lenses move the posture out in space, the subject will localize farther out also. The opposite is true for minus lenses. On the other hand, some psychologists believe that the extraocular muscles relay information to the brain as to the position of the eyes in the orbit. Therefore, can we attribute changes in eye-hand coordination and intersensory localization to accommodation, convergence, or both? With this in mind it was our intention to investigate the above question utilizing the addition of spherical lenses and prisms over the habitually worn prescription of a subject in order to artificially change the accommodation and convergence and then measure the change in intersensory localization. Intersensory localization being the observer\u27s ability to judgementally or behaviourally map one modality on to another

Managing nurse redeployment during the Covid-19 pandemic, lessons for future redeployment: a qualitative study

Background: The mass redeployment of nurses was critical across countries necessitated by the acute health impact of Covid-19. Knowledge was limited regarding how to manage nurse redeployment or the impact that redeployment might have. Redeployment continues, particularly in response to the current staffing crisis and surges such as winter pressures. This study aims to address these gaps in evidence to inform guidance on how best to manage nurse redeployment in practice. Objectives: First, to understand the processes and underpinning decisions made by managers when managing nurse redeployment prior to and during the Covid-19 pandemic. Second, to identify the lessons that can be learned to improve the management of on-going nurse redeployment. Design: Qualitative study utilising semi-structured interviews and focus groups with nurse managers (ISRCTN: 18172749). Setting(s): Three acute National Health Service (NHS) Trusts in England with geographical and ethnic diversity, and different Covid-19 contexts. Participants: Thirty-two nurse managers and four Human Resource advisors responsible for redeploying nurses or receiving and supporting redeployed nurses. Methods: Participants took part in face-to-face or virtual semi-structured interviews from February 2021 to November 2021 and virtual focus groups from July to December 2021. Qualitative data were analysed using reflexive thematic analysis. Results: Four themes were evident in the data, capturing four distinctive phases of the redeployment process. There was a fundamental mismatch between how different parts of the nursing and managerial workforce conceived of their decision-making responsibilities across different phases. This led to managers taking inconsistent and sometimes contradictory approaches when redeploying nurses, and a disconnect between nursing staff at all levels of the chain of command. Furthermore, in conjunction with limited guidance in operationalising redeployment and the distressing experiences vocalised by nurses, nurse managers found nurse redeployment logistically and emotionally challenging; and felt ‘caught in the middle’ of meeting both their managerial and mentoring responsibilities. This became increasingly challenging during subsequent phases of redeployment and remained challenging once the pandemic waned. Conclusions: The approach to nurse redeployment in response to the Covid-19 pandemic prioritised nurse staffing numbers over personal well-being. Key principles of good practice relating to nurse redeployment during the Covid-19 pandemic can be applied to improve future redeployment of nurses and support positive outcomes. Having a planned approach for staff redeployment during normal service delivery comprising operational guidance for those tasked with implementing redeployment, that is scalable in a crisis setting, would be beneficial for the nursing workforce

Profiling essential genes in human mammary cells by multiplex RNAi screening

By virtue of their accumulated genetic alterations, tumor cells may acquire vulnerabilities that create opportunities for therapeutic intervention. We have devised a massively parallel strategy for screening short hairpin RNA (shRNA) collections for stable loss-of-function phenotypes. We assayed from 6000 to 20,000 shRNAs simultaneously to identify genes important for the proliferation and survival of five cell lines derived from human mammary tissue. Lethal shRNAs common to these cell lines targeted many known cell-cycle regulatory networks. Cell line-specific sensitivities to suppression of protein complexes and biological pathways also emerged, and these could be validated by RNA interference (RNAi) and pharmacologically. These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities

Lessons from the frontline: The impact of redeployment during Covid-19 on nurse well-being, performance and retention

Background: Mass redeployment of nurses was critical to the National Health Service response to COVID-19. There remains little understanding of how redeployment was enacted during the pandemic and its impact on nurse managers' and nurses’ mental health and well-being, job performance and retention. This study aimed to understand how nurse redeployment was managed prior to and during COVID-19; explore how nurses made sense of redeployment; and the impact on their mental health and well-being, job performance and retention intentions. Design: A mixed methods approach utilising semistructured interviews, focus groups and surveys with nurse managers and nurses. Setting: Three National Health Service acute hospital trusts. Participants: Thirty-eight nurse managers and human resources advisors participated in interviews and focus groups. Sixty-three nurses who were redeployed or worked with redeployed nurses participated in interviews and surveys over three time points between March 2021 and February 2022. Data collection and analysis: Interviews asked nurse managers about redeployment decisions and nurses about their redeployment experiences. Interview data were analysed using thematic and pen portrait analyses. The survey measured well-being, performance and intentions to leave. Multilevel modelling was conducted to explore relationships between variables over time. Results: Seven themes were identified that illustrate the redeployment process, decisions made, and the impact on nurse managers and nurses. Nurse managers redeployed nurses in response to directives focused on numbers of staff and allowable staff:patient ratios, whereas their decisions were more often person focused. This raised logistical and emotional challenges for nurse managers and a disconnect in the levels of the chain of command regarding the needs of nurses. Most reported feeling like they were treated as a commodity, with redeployment having profound impacts on their mental health, well-being, job performance and retention. The longitudinal pen portrait analysis revealed three ‘journeys’ that represented how nurses made sense of their redeployment, underpinned by two themes: nurse identity and organisational identification. Journeys ranged from those who retained their professional identity and organisational identification (journey one) through to those who experienced a demolition of dual identities (journey three). While most staff in all journeys reported burnout, psychological distress, anxiety, depression and intention to leave their jobs, this was more frequent and severe for those experiencing journey three. These findings, together with stakeholder input, informed the development of 11 recommendations for policy and practice. Limitations: Nurses from minority ethnic backgrounds are under-represented in the sample despite efforts to encourage participation. The quantitative data were planned to be collected at discrete time points during the COVID pandemic for each trust but gaps between data collection time points were compromised by the challenge of ongoing COVID waves and the different set-up times for each trust. Conclusions and future work: Mass redeployment of nurses in response to the COVID-19 pandemic prioritised nurse staffing numbers over staff well-being. Redeployment had a profound impact on nurse managers and nurses with significant and concerning implications reported for nurse well-being, performance and retention. The recommendations for policy and practice will require active endorsement and widespread dissemination and would benefit from evaluation to assess impact. Funding: This synopsis presents independent research funded by the National Institute for Health and Care Research (NIHR) Health and Social Care Delivery Research programme as award number NIHR132041. A plain language summary of this synopsis is available on the NIHR Journals Library Website https://doi.org/10.3310/ EWPE7103

RNAi in cultured mammalian cells using synthetic siRNAs

RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of almost any gene by tapping into innate regulatory mechanisms that are conserved among virtually all eukaryotes. In a typical RNAi experiment, an artificial silencing trigger directs the RNAi pathway toward a target that it would not normally recognize. This is most often an endogenous protein-coding gene, although some noncoding RNAs can also be silenced effectively. The artificial silencing trigger varies; this protocol uses synthetic small interfering RNAs (siRNAs). Lipofectamine 2000 is used to deliver the siRNAs into HEK293 cells. This lipid reagent has proven to be effective for many different cultured mammalian cell lines

Generation of transgenic drosophila expressing shRNAs in the miR-1 backbone

In Drosophila, long-term effects of RNA interference (RNAi) must be achieved by integrating into the genomea template fromwhich anRNAi trigger is transcribed by cellularRNApolymerases, generallyRNA polymerase II or III. With encoded triggers, not only can essentially permanent silencing be achieved, but control can also be exerted over the level of trigger expression, with a resulting variation in the degree to which the target is silenced. Knockdown can also be controlled in a temporal and cell-type-dependent fashionthroughtheuseofwell-establishedtransgenicmethodologiesandwell-testedpromoters.The forms of encoded triggers vary. Long double-stranded RNAs can be expressed as extended inverted repeats. The nearest equivalent of a small interfering RNA is an artificial microRNA (miRNA) or short hairpin RNA (shRNA),whereanaturalmiRNAbackbone(alsocalledascaffold) isremodeledtoproduceadifferent small RNA or a small inverted repeat (<30 nucleotides) is simply expressed. This protocol describes creation of transgenic Drosophila carrying shRNAinserts in a remodeled endogenousmiRNAbackbone.The protocol applies to the use of miRNA-based shRNAs, butmost of the vectors, principles of experimental design, and methods are also applicable to long inverted repeat transgenes

Packaging shRNA retroviruses

To silence a mammalian gene by RNAi using an encoded trigger, a short-hairpin RNA (shRNA) is integrated into the host cell genome as a stable transgene. Target cells are infected with viral plasmid containing shRNA inserted into the vector backbone. Before infection, the plasmid is transfected into a packaging cell line, which provides the trans-acting factors necessary for virus production. These include, minimally, capsid proteins and reverse transcriptase, but they can also include other regulatory factors (e.g., tat for some lentiviral vectors). It is critical to choose the correct packaging cell system for the viral backbone to be used. The packaging cell also defines the host range of the virus, depending on the envelope protein that it expresses. Ecotropic viruses are limited to rodent hosts, whereas amphotropic viruses have a broader host range that also includes humans. Often, investigators will express a nonretroviral envelope, such as vesicular stomatitus virus (VSV) glycoprotein, to enhance virus stability and host range and to enable viruses to be concentrated following production. Although viruses carrying shRNAs are packaged almost identically to viruses carrying protein-encoding genes, one twist is worth noting. shRNAs are efficiently cleaved by the host RNAi biogenesis machinery, which can reduce the level of viral genomic RNAs and consequently viral titers. Therefore, titers can be enhanced by cotransfecting the viral plasmid with a small interfering RNA (siRNA) that targets DGCR- 8/Pasha, which is a core microRNA (miRNA) biogenesis component. siRNAs against Drosha can also be used

Creating an miR30-based shRNA vector

Generating expression constructs for artificial microRNAs (miRNAs) is relatively straightforward. This protocol describes the creation of miR-30-based short hairpin RNA (shRNA) cassettes that are compatible with a number of standard vector systems. The principles outlined here can also be easily applied to other miRNA scaffolds or to simple snapback shRNAs. It is important to note that one must understand the processing of the artificial scaffold and be able to predict precisely the small RNAs that will be generated. Otherwise, no design principles can be effectively applied and the probability that any individual shRNA clone will work effectively will be greatly reduced

Infection of mammalian cells with retroviral shRNAs

Viral infection is a quite simple approach for stably introducing transgenes (e.g., those encoding shorthairpin RNAs [shRNAs]) into the genome. The critical aspects are that the virus and the target cell should be appropriately matched. For example, a virus bearing an ecotropic envelope protein will not infect a human cell line unless the appropriate receptor has been purposefully expressed. VSV-G (vesicular stomatitus virus glycoprotein) pseudotyped viruses have the greatest host range. Nondividing cells can only be infected with lentiviruses, but the additional safety precautions necessary for the use of these tools should dissuade their application to routinely cultured cell lines