Distinct regions of the Swi5 and Ace2 transcription factors are required for specific gene activation

Swi5 and Ace2 are cell cycle-regulated transcription factors that activate expression of early G1-specific genes in Saccharomyces cerevisiae. Swi5 and Ace2 have zinc finger DNA-binding domains that are highly conserved, and the two proteins bind to the same DNA sequences in vitro. Despite this similarity in DNA binding, Swi5 and Ace2 activate different genes in vivo, with Swi5 activating the HO gene and Ace2 activating CTS1 expression. In this report we have used chimeric fusions between Swi5 and Ace2 to determine what regions of these proteins are necessary for promoter-specific activation of HO and CTS1. We have identified specific regions of Swi5 and Ace2 that are required for activation of HO and CTS1, respectively. The Swi5 protein binds HO promoter DNA cooperatively with the Pho2 homeodomain protein, and the HO specificity region of Swi5 identified in the chimeric analysis coincides with the region of Swi5 previously identified that interacts with Pho2 in vitro. Swi5 and Ace2 also activate expression of a number of other genes expressed in G1 phase of the cell cycle, including ASH1, CDC6, EGT2, PCL2, PCL9, RME1, and SIC1. Analysis of the Swi5/Ace2 chimeras shows that distinct regions of Swi5 and Ace2 contribute to the transcriptional activation of some of these other G1-regulated genes

Biotribology of the ageing skin—why we should care

Ageing of populations has emerged as one of the most pressing societal, economic and healthcare challenges currently facing most nations across the globe. The ageing process itself results in degradation of physiological functions and biophysical properties of organs and tissues, and more particularly those of the skin. Moreover, in both developed and emerging economies, population ageing parallels concerning increases in lifestyle-associated conditions such as Type 2 diabetes, obesity and skin cancers. When considered together, these demographic trends call for even greater urgency to find clinical and engineering solutions for the numerous age-related deficits in skin function. From a tribological perspective, detrimental alterations of skin biophysical properties with age have fundamental consequences on how one interacts with the body's inner and outer environments. This stems from the fact that, besides being the largest organ of the human body, and also nearly covering its entirety, the skin is a multifunctional interface which mediates these interactions. The aim of this paper is to present a focused review to discuss some of the consequences of skin ageing from the viewpoint of biotribology, and their implications on health, well-being and human activities. Current and future research questions/challenges associated with biotribology of the ageing skin are outlined. They provide the background and motivation for identifying future lines of research that could be taken up by the biotribology and biophysics communities

Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow

Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed “Bone CLARITY,” a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato–labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue

Systemic RALA/iNOS nanoparticles; a potent gene therapy for metastatic breast cancer coupled as a biomarker of treatment

This study aimed to determine the therapeutic benefit of a nanoparticular formulation for the delivery of inducible nitric oxide synthase (iNOS) gene therapy in a model of breast cancer metastasis. Nanoparticles comprising a cationic peptide vector, RALA, and plasmid DNA were formulated and characterized using a range of physiochemical analyses. Nanoparticles complexed using iNOS plasmids and RALA approximated 60 nm in diameter with a charge of 25 mV. A vector neutralization assay, performed to determine the immunogenicity of nanoparticles in immunocompetent C57BL/6 mice, revealed that no vector neutralization was evident. Nanoparticles harboring iNOS plasmids (constitutively active cytomegalovirus [CMV]-driven or transcriptionally regulated human osteocalcin [hOC]-driven) evoked iNOS protein expression and nitrite accumulation and impaired clonogenicity in the highly aggressive MDA-MB-231 human breast cancer model. Micrometastases of MDA-MB-231-luc-D3H1 cells were established in female BALB/c SCID mice by intracardiac delivery. Nanoparticulate RALA/CMV-iNOS or RALA/hOC-iNOS increased median survival in mice bearing micrometastases by 27% compared with controls and also provoked elevated blood nitrite levels. Additionally, iNOS gene therapy sensitized MDA-MB-231-luc-D3H1 tumors to docetaxel treatment. Studies demonstrated that systemically delivered RALA-iNOS nanoparticles have therapeutic potential for the treatment of metastatic breast cancer. Furthermore, detection of nitrite levels in the blood serves as a reliable biomarker of treatment. Keywords: nonviral gene therapy, nitric oxide, nanoparticle, breast cancer, metastasi

Detection of ctDNA in plasma of patients with clinically localised prostate cancer is associated with rapid disease progression.

BACKGROUND DNA originating from degenerate tumour cells can be detected in the circulation in many tumour types, where it can be used as a marker of disease burden as well as to monitor treatment response. Although circulating tumour DNA (ctDNA) measurement has prognostic/predictive value in metastatic prostate cancer, its utility in localised disease is unknown. METHODS We performed whole-genome sequencing of tumour-normal pairs in eight patients with clinically localised disease undergoing prostatectomy, identifying high confidence genomic aberrations. A bespoke DNA capture and amplification panel against the highest prevalence, highest confidence aberrations for each individual was designed and used to interrogate ctDNA isolated from plasma prospectively obtained pre- and post- (24 h and 6 weeks) surgery. In a separate cohort (n = 189), we identified the presence of ctDNA TP53 mutations in preoperative plasma in a retrospective cohort and determined its association with biochemical- and metastasis-free survival. RESULTS Tumour variants in ctDNA were positively identified pre-treatment in two of eight patients, which in both cases remained detectable postoperatively. Patients with tumour variants in ctDNA had extremely rapid disease recurrence and progression compared to those where variants could not be detected. In terms of aberrations targeted, single nucleotide and structural variants outperformed indels and copy number aberrations. Detection of ctDNA TP53 mutations was associated with a significantly shorter metastasis-free survival (6.2 vs. 9.5 years (HR 2.4; 95% CIs 1.2-4.8, p = 0.014). CONCLUSIONS CtDNA is uncommonly detected in localised prostate cancer, but its presence portends more rapidly progressive disease

"Do I really want to do this?" Longitudinal cohort study participants' perspectives on postal survey design: a qualitative study

<p>Abstract</p> <p>Background</p> <p>Randomised controlled trials have investigated aspects of postal survey design yet cannot elaborate on reasons behind participants' decision making and survey behaviour. This paper reports participants' perspectives of the design of, and participation in, a longitudinal postal cohort survey. It describes strengths and weaknesses in study design from the perspectives of study participants and aims to contribute to the: 1) design of future cohort surveys and questionnaires generally and, 2) design of cohort surveys for people with musculoskeletal disorders (MSDs) specifically.</p> <p>Methods</p> <p>In-depth interviews explored the design of postal surveys previously completed by participants. Interviews used open ended questioning with a topic guide for prompts if areas of interest were not covered spontaneously. Thematic data analysis was undertaken based on the framework method. A second researcher verified all coding.</p> <p>Results</p> <p>Data from fourteen interviews were analysed within three main themes; participation, survey design and survey content. One of the main findings was the importance of clear communication aimed at the correct audience both when inviting potential participants to take part and within the survey itself. Providing enough information about the study, having a topic of interest and an explanation of likely benefits of the study were important when inviting people to participate. The neutrality of the survey and origination from a reputable source were both important; as was an explanation about why information was being collected within the survey itself. Study findings included participants' impressions when invited to take part, why they participated, the acceptability of follow-up of non-responders and why participants completed the follow-up postal survey. Also discussed were participants' first impression of the survey, its length, presentation and participants' views about specific questions within the survey.</p> <p>Conclusions</p> <p>Ideas generated in this study provide an insight into participants' decision making and survey behaviour and may enhance the acceptability of future surveys to potential participants. As well as clear communication, participants valued incentives and survey questions that were relevant to them. However, opinions varied as to the preferred format for responses with some advising more opportunity for open-ended feedback. We also found that some standard format questions can raise quandaries for individual participants.</p

Use of intervention mapping to adapt a health behavior change intervention for endometrial cancer survivors: The shape-up following cancer treatment program

Background: About 80% of endometrial cancer survivors (ECS) are overweight or obese and have sedentary behaviors. Lifestyle behavior interventions are promising for improving dietary and physical activity behaviors, but the constructs associated with their effectiveness are often inadequately reported. The aim of this study was to systematically adapt an evidence-based behavior change program to improve healthy lifestyle behaviors in ECS. Methods: Following a review of the literature, focus groups and interviews were conducted with ECS (n = 16). An intervention mapping protocol was used for the program adaptation, which consisted of six steps: a needs assessment, formulation of matrices of change objectives, selection of theoretical methods and practical applications, program production, adoption and implementation planning, and evaluation planning. Social Cognitive Theory and Control Theory guided the adaptation of the intervention. Results: The process consisted of eight 90-min group sessions focusing on shaping outcome expectations, knowledge, self-efficacy, and goals about healthy eating and physical activity. The adapted performance objectives included establishment of regular eating, balanced diet, and portion sizes, reduction in sedentary behaviors, increase in lifestyle and organized activities, formulation of a discrepancy-reducing feedback loop for all above behaviors, and trigger management. Information on managing fatigue and bowel issues unique to ECS were added. Conclusions: Systematic intervention mapping provided a framework to design a cancer survivor-centered lifestyle intervention. ECS welcomed the intervention and provided essential feedback for its adaptation. The program has been evaluated through a randomized controlled trial