Effects of an Unusual Poison Identify a Lifespan Role for Topoisomerase 2 in Saccharomyces Cerevisiae

A progressive loss of genome maintenance has been implicated as both a cause and consequence of aging. Here we present evidence supporting the hypothesis that an age-associated decay in genome maintenance promotes aging in Saccharomyces cerevisiae (yeast) due to an inability to sense or repair DNA damage by topoisomerase 2 (yTop2). We describe the characterization of LS1, identified in a high throughput screen for small molecules that shorten the replicative lifespan of yeast. LS1 accelerates aging without affecting proliferative growth or viability. Genetic and biochemical criteria reveal LS1 to be a weak Top2 poison. Top2 poisons induce the accumulation of covalent Top2-linked DNA double strand breaks that, if left unrepaired, lead to genome instability and death. LS1 is toxic to cells deficient in homologous recombination, suggesting that the damage it induces is normally mitigated by genome maintenance systems. The essential roles of yTop2 in proliferating cells may come with a fitness trade-off in older cells that are less able to sense or repair yTop2-mediated DNA damage. Consistent with this idea, cells live longer when yTop2 expression levels are reduced. These results identify intrinsic yTop2-mediated DNA damage as a potentially manageable cause of aging

Generation of angular-momentum-dominated electron beams from a photoinjector

Various projects under study require an angular-momentum-dominated electron beam generated by a photoinjector. Some of the proposals directly use the angular-momentum-dominated beams (e.g. electron cooling of heavy ions), while others require the beam to be transformed into a flat beam (e.g. possible electron injectors for light sources and linear colliders). In this paper, we report our experimental study of an angular-momentum-dominated beam produced in a photoinjector, addressing the dependencies of angular momentum on initial conditions. We also briefly discuss the removal of angular momentum. The results of the experiment, carried out at the Fermilab/NICADD Photoinjector Laboratory, are found to be in good agreement with theoretical and numerical models.Comment: 8 pages, 7 figures, submitted to Phys. Rev. ST Accel. Beam

Improved detection of differentially represented DNA barcodes for high-throughput clonal phenomics

Cellular DNA barcoding has become a popular approach to study heterogeneity of cell populations and to identify clones with differential response to cellular stimuli. However, there is a lack of reliable methods for statistical inference of differentially responding clones. Here, we used mixtures of DNA-barcoded cell pools to generate a realistic benchmark read count dataset for modelling a range of outcomes of clone-tracing experiments. By accounting for the statistical properties intrinsic to the DNA barcode read count data, we implemented an improved algorithm that results in a significantly lower false-positive rate, compared to current RNA-seq data analysis algorithms, especially when detecting differentially responding clones in experiments with strong selection pressure. Building on the reliable statistical methodology, we illustrate how multidimensional phenotypic profiling enables one to deconvolute phenotypically distinct clonal subpopulations within a cancer cell line. The mixture control dataset and our analysis results provide a foundation for benchmarking and improving algorithms for clone-tracing experiments

The influence of 1α.25-dihydroxyvitamin D3 coating on implant osseointegration in the rabbit tibia

Objectives: This study aims to evaluate bone response to an implant surface modified by 1α,25-dihydroxyvitamin D3 [1.25-(OH)2D3] in vivo and the potential link between 1.25-(OH) 2D3 surface concentration and bone response. Material and Methods: Twenty-eight implants were divided into 4 groups (1 uncoated control, 3 groups coated with 1.25-(OH)2D3 in concentrations of 10-8, 10-7 and 10-6 M respectively), placed in the rabbit tibia for 6 weeks. Topographical analyses were carried out on coated and uncoated discs using interferometer and atomic-force-microscope (AFM). Twenty-eight implants were histologically observed (bone-to-implant-contact [BIC] and new-bone-area [NBA]). Results: The results showed that the 1.25-(OH)2D3 coated implants presented a tendency to osseointegrate better than the non-coated surfaces, the differences were not significant (P > 0.05). Conclusions: The effect of 1.25-(OH)2D3 coating to implants suggested possible dose dependent effects, however no statistical differences could be found. It is thought that the base substrate topography (turned) could not sustain sufficient amount of 1.25-(OH)2D3 enough to present significant biologic responses. Thus, development a base substrate that can sustain 1.25-(OH)2D3 for a long period is necessary in future studies

Protocol to utilize fresh uncultured human lung tumor cells for personalized functional diagnostics

Peer reviewe

Reciprocal regulation of PKA and rac signaling

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac–bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA–boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac–mediated activation of nuclear Erk1/2 signaling

Functional diagnostics using fresh uncultured lung tumor cells to guide personalized treatments

Functional profiling of a cancer patient's tumor cells holds potential to tailor personalized cancer treatment. Here, we report the utility of fresh uncultured tumor-derived EpCAM(+) epithelial cells (FUTCs) for ex vivo drug response interrogation. Analysis of murine Kras mutant FUTCs demonstrates pharmacological and adaptive signaling profiles comparable to subtype-matched cultured cells. By applying FUTC profiling on non-small cell lung cancer patient samples, we report robust drug-response data in 19 of 20 cases, with cells exhibiting targeted drug sensitivities corresponding to their oncogenic drivers. In one of these cases, an EGFR mutant lung adenocarcinoma patient refractory to osimertinib, FUTC profiling is used to guide compassionate treatment. FUTC profiling identifies selective sensitivity to disulfiram and the combination of carboplatin plus etoposide, and the patient receives substantial clinical benefit from treatment with these agents. We conclude that FUTC profiling provides a robust, rapid, and actionable assessment of personalized cancer treatment options.Peer reviewe

Effect of stress relieving heat treatment on surface topography and dimensional accuracy of incrementally formed grade 1 titanium sheet parts

The forming of parts with an optimized surface roughness and high dimensional accuracy is important in many applications of incremental sheet forming (ISF). To realize this, the effect of stress relieving heat treatment of grade-1 Ti parts performed before and after forming on the surface finish and dimensional accuracy was studied. It was found that heat treatment at a temperature of 540 °C for 2 h improves the surface finish of formed parts resulting in a surface with little or no visible tool marks. Additionally, it improves the dimensional accuracy of parts after unclamping from the rig used for forming, in particular, that of parts with shallow wall angles (typically <25°). It was also noted that post-forming heat treatment improves the surface finish of parts. The surface topography of formed parts was studied using interferometry to yield areal surface roughness parameters and subsequently using secondary electron imaging. Back-scatter electron microscopy imaging results coupled with energy-dispersive X-ray (EDX) analysis showed that heat treatment prior to forming leads to tool wear as indicated by the presence of Fe in samples. Furthermore, post-forming heat treatment prevents curling up of formed parts due to compressive stresses if the formed part is trimmed