Search

Introduction and aims Chronic nonhealing wounds are a life-threatening skin disease in elderly patients and patients with diabetes that costs the National Health Service over £8 billion per year to treat. Unfortunately, current treatments are inadequate, thus there is an urgent need to develop effective therapies. Cellular senescence is emerging as a potential therapeutic target owing to its association with a wide range of age-related pathologies. Cells undergo senescence in response to ageing and stress, losing their ability to proliferate or perform normal tissue functions. Our previous work in mice provided the first mechanistic demonstration that senescent cell accumulation drives poor healing. Moreover, clinical trials currently assessing the efficacy of senescence-targeted drugs (senolytics) are providing repurposing opportunities for cutaneous pathologies. Therefore, the aim of this research is to (i) investigate the role of senescence in human chronic wounds and (ii) determine whether senolytics can be repurposed to promote pathological skin repair. Methods Skin and wounds were collected from healthy, aged and diabetic donors to quantify senescence levels (senescence-associated β-galactosidase), characterize senescent cell types (immunofluorescence) and determine key drivers of skin senescence (RNA sequencing). Additionally, we screened the wound healing potential of a panel of senolytics in healthy skin vs. skin from patients with chronic wounds. Results We found significant accumulation of senescence in pathological skin wounds, localized to stromal cells. Senescence levels also correlated substantially with clinical parameters, such as wound site, diabetic status and infection. RNA sequencing revealed key links between senescence and immunity, suggesting inflammation as a crucial driver of skin senescence. Finally, senolytic treatment significantly accelerated healing in skin from patients with chronic wounds. Conclusions Our data provide the first in-depth characterization of senescence in human chronic skin wounds, crucially identifying novel therapeutic targets of senescence, while simultaneously assessing the potential of drug repurposing for patient benefit. [ABSTRACT FROM AUTHOR]

Rhodotorula (Rhodosporidium) toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70% of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3, and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides. [ABSTRACT FROM AUTHOR]

Risk and safety programs need to be monitored and evaluated through real-world experience and performance. Several perspectives on the program might be useful across organizational units, stakeholders, and subject-matter experts. Quantifications of risks, benefits, and costs of the programs as well as the associated parameter uncertainties will vary by perspective. The literature has quantified the benefits of a safety program in single stakeholder/expert perspectives. This paper describes a layering of perspectives of program effectiveness and characterizes the variation of estimated program efficacy across the several perspectives. Within each perspective, the uncertainties of model selection and the estimation of cost-benefit analysis parameters are addressed via interval numbers. The approach is demonstrated for safety trainings that are aimed to reduce runway incursions for 80 airports across a region, an issue of highest priority for the US National Transportation Safety Board. The several perspectives of the demonstration are the pilot/driver, the airport owner/operator, and the safety regulator. The results suggest for each of the perspectives which of the airports should receive the trainings. The paper will be of interest for the performance evaluation of safety programs with uncertainties of program benefits and costs and multiple agencies, users, customers, and other stakeholders/experts. [ABSTRACT FROM AUTHOR]

Background: The exacting nutritional requirements and complicated life cycles of parasites mean that they are not always amenable to high-throughput drug screening using automated procedures. Therefore, we have engineered the yeast Saccharomyces cerevisiae to act as a surrogate for expressing anti-parasitic targets from a range of biomedically important pathogens, to facilitate the rapid identification of new therapeutic agents. Methodology/Principal Findings: Using pyrimethamine/dihydrofolate reductase (DHFR) as a model parasite drug/drug target system, we explore the potential of engineered yeast strains (expressing DHFR enzymes from Plasmodium falciparum, P. vivax, Homo sapiens, Schistosoma mansoni, Leishmania major, Trypanosoma brucei and T. cruzi) to exhibit appropriate differential sensitivity to pyrimethamine. Here, we demonstrate that yeast strains (lacking the major drug efflux pump, Pdr5p) expressing yeast (ScDFR1), human (HsDHFR), Schistosoma (SmDHFR), and Trypanosoma (TbDHFR and TcDHFR) DHFRs are insensitive to pyrimethamine treatment, whereas yeast strains producing Plasmodium (PfDHFR and PvDHFR) DHFRs are hypersensitive. Reassuringly, yeast strains expressing field-verified, drug-resistant mutants of P. falciparum DHFR (Pfdhfr51I,59R,108N) are completely insensitive to pyrimethamine, further validating our approach to drug screening. We further show the versatility of the approach by replacing yeast essential genes with other potential drug targets, namely phosphoglycerate kinases (PGKs) and N-myristoyl transferases (NMTs). Conclusions/Significance: We have generated a number of yeast strains that can be successfully harnessed for the rapid and selective identification of urgently needed anti-parasitic agents. Author Summary: Parasites kill millions of people every year and leave countless others with chronic debilitating disease. These diseases, which include malaria and sleeping sickness, mainly affect people in developing countries. For this reason, few drugs have been developed to treat them. To make matters worse, many parasites are developing resistance to the drugs that are available. Thus, there is an urgent need to develop new drugs, but this is hampered by the fact that most parasites are difficult or impossible to grow in the laboratory. To address this, we have engineered baker's yeast to be dependent on the function of enzymes from either parasites or humans. In all, our engineered yeast constructs encompass six parasites (causing malaria, schistosomiasis, leishmaniasis, sleeping sickness, and Chagas disease) and three different enzymes that are known or potential drug targets. Further, we have increased yeast's sensitivity to drugs by deleting the gene for its major drug efflux pump. Because yeast is robust and easy to grow in the laboratory, we can use a robot to screen for drugs that will kill yeast dependent on a parasite enzyme, but not touch yeast dependent on the equivalent human enzyme. [ABSTRACT FROM AUTHOR]