An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence

Background: Cellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism.Results: To identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells.Conclusion: The senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-kappa B signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action

The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state

Influence of a knot on the strength of a polymer strand

Many experiments have been done to determine the relative strength of different knots, and these show that the break in a knotted rope almost invariably occurs at a point just outside the `entrance' to the knot. The influence of knots on the properties of polymers has become of great interest, in part because of their effect on mechanical properties. Knot theory applied to the topology of macromolecules indicates that the simple trefoil or `overhand' knot is likely to be present with high probability in any long polymer strand. Fragments of DNA have been observed to contain such knots in experiments and computer simulations. Here we use {\it ab initio} computational methods to investigate the effect of a trefoil knot on the breaking strength of a polymer strand. We find that the knot weakens the strand significantly, and that, like a knotted rope, it breaks under tension at the entrance to the knot.Comment: 3 pages, 4 figure

Clinical indications for digital imaging in dento-alveolar trauma. Part 1: traumatic injuries

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73819/1/j.1600-9657.2006.00509.x.pd

Characterization of linewidth variation on 248- and 193-nm exposure tools

Proceedings of S P I E - the International Society for OpticalThe line-width variation of a 193 nm lithographic process utilizing a 0.60 NA scanner and a binary reticle is compared to that of a 248 nm lithographic processes utilizing a 0.68 NA scanner and a variety of reticle technologies. These include binary, attenuated PSM with assist features and alternating PSM reticles. Despite the fact that the 193 nm tool has a lower NA and that the data was generated using a binary reticle, the 193 nm lithographic process allows for the line-width values to be pushed lower than previously achieved with 248 nm lithographic processes. The 3-sigma values from 4000 electrical linewidth measurements per wafer (160 measurements per 25*25 mm field, 25 fields per wafer) were calculated for different mask features. The 193 nm process was capable of reaching line-widths needed for future generations of advance logic chips. Compared to the 193 nm process utilizing a binary reticle, only the 248 nm processes utilizing either an attenuated PSM with assist features or an alternating PSM reticle had similarly low line-width variation. The 248 nm processes utilizing a binary reticle had higher line-width variation even at larger poly gate conductor line-widths.published_or_final_versio

Temperature- and thickness-dependent elastic moduli of polymer thin films

The mechanical properties of polymer ultrathin films are usually different from those of their counterparts in bulk. Understanding the effect of thickness on the mechanical properties of these films is crucial for their applications. However, it is a great challenge to measure their elastic modulus experimentally with in situ heating. In this study, a thermodynamic model for temperature- (T) and thickness (h)-dependent elastic moduli of polymer thin films Ef(T,h) is developed with verification by the reported experimental data on polystyrene (PS) thin films. For the PS thin films on a passivated substrate, Ef(T,h) decreases with the decreasing film thickness, when h is less than 60 nm at ambient temperature. However, the onset thickness (h*), at which thickness Ef(T,h) deviates from the bulk value, can be modulated by T. h* becomes larger at higher T because of the depression of the quenching depth, which determines the thickness of the surface layer δ

The study protocol of a cluster-randomised controlled trial of family-mediated personalised activities for nursing home residents with dementia

<p>Abstract</p> <p>Background</p> <p>Following admission to a nursing home, the feelings of depression and burden that family carers may experience do not necessarily diminish. Additionally, they may experience feelings of guilt and grief for the loss of a previously close relationship. At the same time, individuals with dementia may develop symptoms of depression and agitation (BPSD) that may be related to changes in family relationships, social interaction and stimulation. Until now, interventions to alleviate carer stress and BPSD have treated carers and relatives separately rather than focusing on maintaining or enhancing their relationships. One-to-one structured activities have been shown to reduce BPSD and also improve the caring experience, but barriers such as a lack of resources impede the implementation of activities in aged care facilities. The current study will investigate the effect of individualised activities based on the Montessori methodology administered by family carers in residential care.</p> <p>Methods/Design</p> <p>We will conduct a cluster-randomised trial to train family carers in conducting personalised one-to-one activities based on the Montessori methodology with their relatives. Montessori activities derive from the principles espoused by Maria Montessori and subsequent educational theorists to promote engagement in learning, namely task breakdown, guided repetition, progression in difficulty from simple to complex, and the careful matching of demands to levels of competence. Persons with dementia living in aged care facilities and frequently visiting family carers will be included in the study. Consented, willing participants will be randomly assigned by facility to a treatment condition using the Montessori approach or a control waiting list condition. We hypothesise that family carers conducting Montessori-based activities will experience improvements in quality of visits and overall relationship with the resident as well as higher self-rated mastery, fewer depressive symptoms, and a better quality of life than carers in the waiting list condition.</p> <p>Discussion</p> <p>We hypothesise that training family carers to deliver personalised activities to their relatives in a residential setting will make visits more satisfying and may consequently improve the quality of life for carers and their relatives. These beneficial effects might also reduce nursing staff burden and thus impact positively on residential facilities.</p> <p>Trial Registration</p> <p>Australian New Zealand Clinical Trials Registry - <a href="http://www.anzctr.org.au/ACTRN12611000998943.aspx">ACTRN12611000998943</a></p

NK-, NKT-and CD8-derived IFNγ drives myeloid cell activation and erythrophagocytosis, resulting in Trypanosomosis-associated acute anemia

African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia

A feasibility study comparing UK older adult mental health inpatient wards which use protected engagement time with other wards which do not: Study protocol

© 2016 Nolan et al. Background: Protected engagement time (PET) is a concept of managing staff time on mental health inpatient wards with the aim of increasing staff and patient interaction. Despite apparent widespread use of PET, there remains a dearth of evidence as to how it is implemented and whether it carries benefits for staff or patients. This protocol describes a study which is being carried out on mental health wards caring for older adults (aged over 65) in England. The study shares a large proportion of the procedures, measures and study team membership of a recently completed investigation of the impact of PET in adult acute mental health wards. The study aims to identify prevalence and components of PET to construct a model for the intervention, in addition to testing the feasibility of the measures and procedures in preparation for a randomised trial. Methods/design: The study comprises four modules and uses a mixed methods approach. Module 1 involves mapping all inpatient wards in England which provide care for older adults, including those with dementia, ascertaining how many of these provide PET and in what way. Module 2 uses a prospective cohort method to compare five older adult mental health wards that use PET with five that do not across three National Health Service (NHS) Foundation Trust sites. The comparison comprises questionnaires, observation tools and routinely collected clinical service data and combines validated measures with questions developed specifically for the study. Module 3 entails an in-depth case study evaluation of three of the participating PET wards (one from each NHS Trust site) using semi-structured interviews with patients, carers and staff. Module 4 describes the development of a model and fidelity scale for PET using the information derived from the other modules with a working group of patients, carers and staff. Discussion: This is a feasibility study to test the application of the measures and methods in inpatient wards for older adults and develop a draft model for the intervention. The next stage will prospectively involve testing of the model and fidelity scale in randomised conditions to provide evidence for the effectiveness of PET as an intervention