Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching

A major hallmark of Alzheimer’s disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1–42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions

Monocoque structure for the SKITTER three-legged walker

The SKITTER 2 design is a monocoque version of the proposed lunar three-legged walker. By the definition of monocoque, the body and legs are a shell with no internal ribbing or supports added for absorbing stresses. The purpose of the monocoque is to encase the elements used for power transmission, power supply, and control of the motion. The material for the structure is a vinyl ester resin, Derakane 8084. This material is easily formable and locally obtainable. The body consists of a hexagonally shaped cylinder with truncated hexagonal pyramids on the top and botton. The legs are eight inch diameter cylinders. The legs are comprised of a tibia section and a femur section. The SKITTER 2 is powered by six actuators which provide linear forces that are transformed into rotary torques by a series of chains and sprockets. The joints connect the femur to the body and the tibia to the femur. Surrounding the joints are flexible rubber hoses that fully encase the chains and sprockets. The SKITTER 2 is capable of walking upside down, righting itself after being overturned, and has the ability to perform in many environments. Applications for this walker include lunar transport or drilling, undersea exploration, and operation in severe surroundings such as arctic temperatures or high radiation

Loss of 5-hydroxymethylcytosine correlates with increasing morphologic dysplasia in melanocytic tumors

DNA methylation is the most well studied epigenetic modification in cancer biology. 5-hydroxymethylcytosine is an epigenetic mark that can be converted from 5-methylcytosine by the ten-eleven translocation gene family. We recently reported the loss of 5-hydroxymethylcytosine in melanoma compared to benign nevi and suggested that loss of this epigenetic marker is correlated with tumor virulence based on its association with a worse prognosis. In this study we further characterize the immunoreactivity patterns of 5-hydroxymethylcytosine in the full spectrum of melanocytic lesions to further validate the potential practical application of this epigenetic marker. 175 cases were evaluated: 18 benign nevi, 20 dysplastic nevi (10 low-grade and 10 high-grade lesions), 10 atypical Spitz nevi, 20 borderline tumors, 5 melanomas arising within nevi, and 102 primary melanomas. Progressive loss of 5-hydroxymethylcytosine from benign dermal nevi to high-grade dysplastic nevi to borderline melanocytic neoplasms to melanoma was observed. In addition, an analysis of the relationship of nuclear diameter to 5-hydroxymethylcytosine staining intensity within lesional cells revealed a significant correlation between larger nuclear diameter and decreased levels of 5-hydroxymethylcytosine. Furthermore, borderline lesions uniquely exhibited a diverse spectrum of staining of each individual case. This study further substantiates the association of 5-hydroxymethylcytosine loss with dysplastic cytomorphologic features and tumor progression and supports the classification of borderline lesions as a biologically distinct category of melanocytic lesions

The Role of I Region Gene Products in Macrophage - T Lymphocyte Interaction

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73965/1/j.1600-065X.1978.tb00400.x.pd

Clinical and genetic determinants of warfarin pharmacokinetics and pharmacodynamics during treatment initiation

Variable warfarin response during treatment initiation poses a significant challenge to providing optimal anticoagulation therapy. We investigated the determinants of initial warfarin response in a cohort of 167 patients. During the first nine days of treatment with pharmacogenetics-guided dosing, S-warfarin plasma levels and international normalized ratio were obtained to serve as inputs to a pharmacokinetic-pharmacodynamic (PK-PD) model. Individual PK (S-warfarin clearance) and PD (I max) parameter values were estimated. Regression analysis demonstrated that CYP2C9 genotype, kidney function, and gender were independent determinants of S-warfarin clearance. The values for I max were dependent on VKORC1 and CYP4F2 genotypes, vitamin K status (as measured by plasma concentrations of proteins induced by vitamin K absence, PIVKA-II) and weight. Importantly, indication for warfarin was a major independent determinant of I max during initiation, where PD sensitivity was greater in atrial fibrillation than venous thromboembolism. To demonstrate the utility of the global PK-PD model, we compared the predicted initial anticoagulation responses with previously established warfarin dosing algorithms. These insights and modeling approaches have application to personalized warfarin therapy. © 2011 Gong et al

Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers

Should Research Ethics Encourage the Production of Cost-Effective Interventions?

This project considers whether and how research ethics can contribute to the provision of cost-effective medical interventions. Clinical research ethics represents an underexplored context for the promotion of cost-effectiveness. In particular, although scholars have recently argued that research on less-expensive, less-effective interventions can be ethical, there has been little or no discussion of whether ethical considerations justify curtailing research on more expensive, more effective interventions. Yet considering cost-effectiveness at the research stage can help ensure that scarce resources such as tissue samples or limited subject popula- tions are employed where they do the most good; can support parallel efforts by providers and insurers to promote cost-effectiveness; and can ensure that research has social value and benefits subjects. I discuss and rebut potential objections to the consideration of cost-effectiveness in research, including the difficulty of predicting effectiveness and cost at the research stage, concerns about limitations in cost-effectiveness analysis, and worries about overly limiting researchers’ freedom. I then consider the advantages and disadvantages of having certain participants in the research enterprise, including IRBs, advisory committees, sponsors, investigators, and subjects, consider cost-effectiveness. The project concludes by qualifiedly endorsing the consideration of cost-effectiveness at the research stage. While incorporating cost-effectiveness considerations into the ethical evaluation of human subjects research will not on its own ensure that the health care system realizes cost-effectiveness goals, doing so nonetheless represents an important part of a broader effort to control rising medical costs

Meiotic Chromosome Pairing Is Promoted by Telomere-Led Chromosome Movements Independent of Bouquet Formation

Chromosome pairing in meiotic prophase is a prerequisite for the high fidelity of chromosome segregation that haploidizes the genome prior to gamete formation. In the budding yeast Saccharomyces cerevisiae, as in most multicellular eukaryotes, homologous pairing at the cytological level reflects the contemporaneous search for homology at the molecular level, where DNA double-strand broken ends find and interact with templates for repair on homologous chromosomes. Synapsis (synaptonemal complex formation) stabilizes pairing and supports DNA repair. The bouquet stage, where telomeres have formed a transient single cluster early in meiotic prophase, and telomere-promoted rapid meiotic prophase chromosome movements (RPMs) are prominent temporal correlates of pairing and synapsis. The bouquet has long been thought to contribute to the kinetics of pairing, but the individual roles of bouquet and RPMs are difficult to assess because of common dependencies. For example, in budding yeast RPMs and bouquet both require the broadly conserved SUN protein Mps3 as well as Ndj1 and Csm4, which link telomeres to the cytoskeleton through the intact nuclear envelope. We find that mutants in these genes provide a graded series of RPM activity: wild-type>mps3-dCC>mps3-dAR>ndj1Δ>mps3-dNT = csm4Δ. Pairing rates are directly correlated with RPM activity even though only wild-type forms a bouquet, suggesting that RPMs promote homologous pairing directly while the bouquet plays at most a minor role in Saccharomyces cerevisiae. A new collision trap assay demonstrates that RPMs generate homologous and heterologous chromosome collisions in or before the earliest stages of prophase, suggesting that RPMs contribute to pairing by stirring the nuclear contents to aid the recombination-mediated homology search