A Genealogy of Autonomy: Freedom, Paternalism, and the Future of the Doctor-Patient Relationship.

Although the principle of respect for personal autonomy has been the subject of debate for almost 40 years, the conversation has often suffered from lack of clarity regarding the philosophical traditions underlying this principle. In this article, I trace a genealogy of autonomy, first contrasting Kant's autonomy as moral obligation and Mill's teleological political liberty. I then show development from Mill's concept to Beauchamp and Childress' principle and to Julian Savulescu's non-teleological autonomy sketch. I argue that, although the reach for a new principle to guide choices in physician-patient relationships can rightfully be seen as important, the notion that is now called autonomy within bioethics has corollaries that undermine critical aspects of medical care. As such, there is need for a richer account of the interplay between the free choice of patients and the informed recommendations of doctors.

Dignity reevaluated: A theological examination of human dignity and the role of the Church in bioethics and end-of-life care.

Predominant among the terminological ambiguities that plague contemporary bioethics is confusion attending the meaning of the term "human dignity," particularly as it applies to so-called end-of-life discussions. This study surveys current trends in treatment of the concept of dignity, examining relevant thinkers who see dignity as redundant or as capability-dependent. These inadequate views are contrasted with an attitude, based theologically in Mark 5, that understands human dignity to represent an absolute characteristic that is donated graciously to all bearers of imago Dei. Human dignity must thus be affirmed as inviolable and independent of autonomy, rationality, or capability. A specific task of the Christian Church is to faithfully recognize and proclaim this dignity. This investigation is particularly relevant in the face of contemporary discussions regarding euthanasia and physician-assisted death. Lay Summary: Much of the polarization within the contemporary bioethical discussion proceeds out of confusion regarding how we ought to define the terms of the debate. If we may take the existing debates regarding euthanasia and physician-assisted death as an example, we may understand the vital need for a sensible definition of the term that stands at the heart of the arguments of both sides of the debate: "human dignity." As such, this study seeks to define dignity in a logical, theological, deeply personal, and highly practical fashion, and to outline the critical role of the Church within such an understanding. Sometimes, when I walk into the room, he ignores me. Sometimes he thinks I am someone else. Most often he is asleep, subjugated by drugs designed to prevent agitation, although "agitation" is the sterilized hospital word for what I would call unbridled panic, total disorientation. The night he had the stroke, they had to tie him to the bed just to keep him in the hospital. And they wouldn't let me see him because he had been calling my name. Very dramatic, but most of his suffering, and our grief, is not dramatic. It is just the mundane process of one man slowly fading, becoming, every day, more of a stranger to himself and to those he loves.

Total Synthesis of Ustiloxin D Utilizing an Ammonia-Ugi Reaction.

Total synthesis of the highly functionalized cyclic peptide natural product, ustiloxin D, has been achieved in a convergent manner. Our strategy incorporates an asymmetric allylic alkylation to construct the tert-alkyl aryl ether linkage between the dopa and isoleucine residues. The elaborated ß-hydroxydopa derivative is rapidly converted to a linear tripeptide through an ammonia-Ugi reaction. Subsequent cyclization and global deprotection affords ustiloxin D in six steps from a known ß-hydroxydopa derivative.

A general method for interconversion of boronic acid protecting groups: trifluoroborates as common intermediates.

We have developed a general protocol for the interconversion of diverse protected boronic acids, via intermediate organotrifluoroborates. N-Methyliminodiacetyl boronates, which have been hitherto resistant to direct conversion to trifluoroborates, have been shown to undergo fluorolysis at elevated temperatures. Subsequent solvolysis of organotrifluoroborates in the presence of trimethylsilyl chloride and a wide range of bis-nucleophiles enables the generation of a variety of protected boronic acids.

Naturally occurring polyphenolic inhibitors of amyloid beta aggregation.

Alzheimer's disease is the most common neurodegenerative disease and is one of the main causes of death in developed countries. Consumption of foods rich in polyphenolics is strongly correlated with reduced incidence of Alzheimer's disease. Our study has investigated the biological activity of previously untested polyphenolic compounds in preventing amyloid ß aggregation. The anti-aggregatory potential of these compounds was assessed using the Thioflavin-T assay, transmission electron microscopy, dynamic light scattering and size exclusion chromatography. Two structurally related compounds, luteolin and transilitin were identified as potent inhibitors of Aß fibril formation. Computational docking studies with an X-ray derived oligomeric structure offer a rationale for the inhibitory activity observed and may facilitate development of improved inhibitors of Aß aggregation and toxicity.

NvPrdm14d-expressing neural progenitor cells contribute to non-ectodermal neurogenesis in Nematostella vectensis.

Neurogenesis has been studied extensively in the ectoderm, from which most animals generate the majority of their neurons. Neurogenesis from non-ectodermal tissue is, in contrast, poorly understood. Here we use the cnidarian Nematostella vectensis as a model to provide new insights into the molecular regulation of non-ectodermal neurogenesis. We show that the transcription factor NvPrdm14d is expressed in a subpopulation of NvSoxB(2)-expressing endodermal progenitor cells and their NvPOU4-expressing progeny. Using a new transgenic reporter line, we show that NvPrdm14d-expressing cells give rise to neurons in the body wall and in close vicinity of the longitudinal retractor muscles. RNA-sequencing of NvPrdm14d::GFP-expressing cells and gene knockdown experiments provide candidate genes for the development and function of these neurons. Together, the identification of a population of endoderm-specific neural progenitor cells and of previously undescribed putative motoneurons in Nematostella provide new insights into the regulation of non-ectodermal neurogenesis.

The Notch pathway in the annelid Platynereis: insights into chaetogenesis and neurogenesis processes.

Notch is a key signalling pathway playing multiple and varied functions during development. Notch regulates the selection of cells with a neurogenic fate and maintains a pool of yet uncommitted precursors through lateral inhibition, both in insects and in vertebrates. Here, we explore the functions of Notch in the annelid Platynereis dumerilii (Lophotrochozoa). Conserved components of the pathway are identified and a scenario for their evolution in metazoans is proposed. Unexpectedly, neither Notch nor its ligands are expressed in the neurogenic epithelia of the larva at the time when massive neurogenesis begins. Using chemical inhibitors and neural markers, we demonstrate that Notch plays no major role in the general neurogenesis of larvae. Instead, we find Notch components expressed in nascent chaetal sacs, the organs that produce the annelid bristles. Impairing Notch signalling induces defects in chaetal sac formation, abnormalities in chaetae producing cells and a change of identity of chaeta growth accessory cells. This is the first bilaterian species in which the early neurogenesis processes appear to occur without a major involvement of the Notch pathway. Instead, Notch is co-opted to pattern annelid-specific organs, likely through a lateral inhibition process. These features reinforce the view that Notch signalling has been recruited multiple times in evolution due to its remarkable 'toolkit' nature.

Iron, Copper, and Zinc Concentration in Aß Plaques in the APP/PS1 Mouse Model of Alzheimer's Disease Correlates with Metal Levels in the Surrounding Neuropil.

The metal ions of iron, copper, and zinc have long been associated with the aggregation of ß-amyloid (Aß) plaques in Alzheimer's disease; an interaction that has been suggested to promote increased oxidative stress and neuronal dysfunction. We examined plaque metal load in the hippocampus of APP/PS1 mice using X-ray fluorescence microscopy to assess how the anatomical location of Aß plaques was influenced by the metal content of surrounding tissue. Immunohistochemical staining of Aß plaques colocalized with areas of increased X-ray scattering power in unstained tissue sections, allowing direct X-ray based-assessment of plaque metal levels in sections subjected to minimal chemical fixation. We identified and mapped 48 individual plaques in four subregions of the hippocampus from four biological replicates. Iron, Cu, and Zn areal concentrations (ng cm-2) were increased in plaques compared to the surrounding neuropil. However, this elevation in metal load reflected the local metal makeup of the surrounding neuropil, where different brain regions are enriched for different metal ions. After correcting for tissue density, only Zn levels remained elevated in plaques. This study suggests that the in vivo binding of Zn to plaques is not simply due to increased protein deposition.

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis.

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

Therapeutic targeting and rapid mobilization of endosteal HSC using a small molecule integrin antagonist.

The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules. Here, we show targeting α9ß1/α4ß1 integrins with a single dose of a small molecule antagonist (BOP (N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine)) rapidly mobilizes long-term multi-lineage reconstituting HSC. Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100. Impressively, HSC in equal volumes of peripheral blood (PB) mobilized with this combination effectively out-competes PB mobilized with G-CSF. The enhanced mobilization observed using BOP and AMD3100 is recapitulated in a humanized NODSCIDIL2Rγ(-/-) model, demonstrated by a significant increase in PB CD34(+) cells. Using a related fluorescent analogue of BOP (R-BC154), we show that this class of antagonists preferentially bind human and mouse HSC and progenitors via endogenously primed/activated α9ß1/α4ß1 within the endosteal niche. These results support using dual α9ß1/α4ß1 inhibitors as effective, rapid and transient mobilization agents with promising clinical applications.

Expression of the CEA gene family members NCA-50/90 and NCA-160 (CD66) in childhood acute lymphoblastic leukemias (ALLs) and in cell lines of B-cell origin.

The carcinoembryonic antigen (CEA) and the classical non-specific cross-reacting antigens (NCAs) belong to the CEA gene family which is part of the immunoglobulin superfamily. In normal hematopoiesis, CEA gene family members (CGMs) have only been reported on cells of myeloid and monocytic origin. In the present study, we analyzed 62 childhood acute lymphoblastic leukemias (ALLs) and seven surface immunoglobulin positive (sig+) B-cell lines for the expression of the CEA family members CEA, NCA-50/90, NCA-95, NCA-160, CGM1 and CGM7. We demonstrated that members of the CEA family were present in 76% of childhood ALLs of B- and T-cell origin. In ALLs of B-cell origin, 82% of the samples expressed at least one CEA subgroup member: 38% NCA-50/90 (CD66c), 31% NCA-160 (CD66a), and 13% both. Six of seven B-cell lines solely expressed NCA-160. In seven ALL of T-cell origin, sole NCA-160 expression was present in 29% of the cases. CEA and CGM1 were not expressed in childhood ALLs or in the sIg+ B-cell lines. In 15 ALLs and seven B-cell lines which could be analyzed for CGM7 expression, the antigen was not detected. NCA-95 was not expressed in 91% of the B-lineage ALLs, in T-lineage ALLs and in the B-cell lines. However, five B-lineage ALLs showed conflicting data on the binding patterns of two, on leukocytes specifically NCA-95 recognizing antibodies suggesting either expression of unknown forms of NCA-95 or NCA-50/90 or of a yet unknown member of the CEA family in these ALL cells. The expression of CEA subgroup members in childhood ALL cells might have prognostic impacts, as an inverse correlation exists between NCA expression on leukemic blasts and the risk factor white blood count at diagnosis.

Apoptosis by 6-O-palmitoyl-L-ascorbic acid coincides with JNK-phosphorylation and inhibition of Mg2+-dependent phosphatase activity.

6-O-Palmitoyl ascorbic acid (PAA) has recently been used as a substitute for ascorbic acid because of its greater potency as an antioxidant. In detailed concentration response studies distinct cytotoxic effects of PAA at concentrations exceeding 100 microM were reported. Here we examined and further characterized this cytotoxicity. While ascorbic acid was tolerated well up to millimolar concentrations, PAA revealed an LC50 between 125 and 150 microM in rat GH3 tumor cells. Morphological and biochemical observations suggested the induction of apoptosis at concentrations exceeding 125 microM with a prominent activation of caspase 3 at 250 microM after 4 hr. A subsequent pronounced fragmentation of DNA (DNA-ladder) was detected after 6 hr and was further enhanced after 12 hr. The activation of caspases and the cleavage of its substrate PARP was preceded by a distinct increase in the phosphorylation of stress activated JNK-kinases. This observation suggested that the agent affected signal transduction mechanisms regulating protein phosphorylation at serine/threonine residues in the cell. No effect of PAA on protein phosphatase 2A (PP2A)-like activity was observed while magnesium-dependent protein phosphatase activity, presumably PP2C, was inhibited concentration-dependently up to 75% at the respective concentrations. Thus, the cytotoxic, pro-apoptotic effect of PAA might be related to the inhibition of PP2C and the activation of JNK.

Characterization of two pituitary GH3 cell sublines partially resistant to apoptosis induction by okadaic acid.

Pituitary GH3 cells die by apoptosis when treated with okadaic acid, a specific inhibitor of ser/thr phosphatases. Incubations starting at concentrations of 5 and 12.5 nM followed by stepwise rises resulted in two populations (the S1 and S2 sublines) that proliferated at initially lethal 30 nM. Cells were partially resistant to higher concentrations of okadaic acid and its derivative methyl okadaate. Toxicity of the structurally distinct inhibitors cantharidic acid and calyculin A was differently affected in the two resistant lines. The enhanced expression of the P-glycoprotein was one mechanism of resistance in S1 and S2. Resistance was reversed completely (S1) or partially (S2) by the addition of verapamil. In addition, phosphatase activity, presumably PP2A, was increased in S2. Therefore, pharmacokinetic and pharmacodynamic mechanisms can protect pituitary GH3 cells from apoptotic cell death by okadaic acid.

Inhibitors of ser/thr phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells.

Two structurally different inhibitors of ser/thr phosphatases 1 and 2A, okadaic acid and calyculin A, time- and concentration-dependently stimulated and inhibited cell-specific function (hormone gene expression) in pituitary GH3 cells. The negative effect was associated with the appearance of apoptotic cell death. Nanomolar concentrations of both agents produced the characteristic morphological alterations and a DNA fragmentation ladder. Calyculin A treatment resulted in comparable changes with 10fold lower concentrations than okadaic acid. Observations with derivatives of okadaic acid with no or lower phosphatase inhibitory potency supported the conclusion that apoptosis induction is related to inhibition of ser/thr phosphatases, presumably types 1 and 2A. Membrane damage as measured by lactate dehydrogenase liberation into medium was significantly lower in apoptotic vs. necrotic cells. DNA fragmentation could be reduced by the addition of zinc but not by removal of extracellular calcium with EGTA. Apoptotic changes were reduced by the concomitant activation of protein kinase A by a membrane permeable cAMP analogue. Incubation of cells for 4 months in successively increased concentrations of okadaic acid resulted in a population that proliferated at the initially lethal concentration of 30 nM.

Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells.

Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.

A fast and sensitive technique to study the kinetics and the concentration dependencies of DNA fragmentation during drug-induced apoptosis.

Apoptotic cell death with its characteristic coordinated cellular breakdown can be triggered by cytotoxic drugs. One prominent feature that differentiates apoptotic from necrotic cell death is the caspase-mediated activation of an endonuclease that internucleosomally cleaves DNA resulting in the so-called apoptotic DNA ladder. Here we report a new rapid, sensitive and inexpensive column separation technique to study drug-induced DNA fragmentation from 10(6) or less cells. This technique, which is based on a modified plasmid spin column kit, avoids the use of hazardous chemicals. With this procedure and subsequent densitometric analysis it was possible to study the concentration dependencies and the kinetics of drug-induced DNA fragmentation. The applicability of this technique is shown for okadaic acid- and cantharidic-acid-treated pituitary GH(3) cells as well as highly okadaic-acid-resistant sublines. These studies allowed us to compare as well as to differentiate the effects and potencies of these structurally different but functionally quite similar inhibitors of ser/thr phosphatases 1 and 2A.

Phlebotomy treatment for elimination of perfluoroalkyl acids in a highly exposed family: a retrospective case-series.

BACKGROUND: Perfluoroalkyl acids (PFAAs) are a family of commonly used synthetic chemicals that have become widespread environmental contaminants. In human serum, perfluorohexane sulfonate (PFHxS), perflurooctane sulfonate (PFOS), and perfluorooctanoate (PFOA) are most frequently detected, in part owing to their long elimination half-lives of between 3.8 yrs (PFOA) and 8.5 yrs (PFHxS). These PFAAs also cross the placenta and have been associated with developmental toxicity, and some are considered likely human carcinogens. Interventions to eliminate PFAAs in highly contaminated individuals would reduce future health risks, but minimal research has been conducted on methods to facilitate accelerated human clearance of these persistent substances. METHODS: Six patients with elevated serum concentrations from a single family were treated by intermittent phlebotomy over a 4-5 year period at intervals similar to, or less frequent than what is done for routine blood donation at Canadian Blood Services. The apparent elimination half-life (HLapp) for PFHxS, PFOS, and PFOA in this treated population was calculated in each patient and compared to the intrinsic elimination half-lives (HLin) from a literature reference population of untreated fluorochemical manufacturing plant retirees (n = 26, age >55 yrs). RESULTS: For all three PFAAs monitored during phlebotomy, HLapp in each of the family members (except the mother, who had a low rate of venesection) was significantly shorter than the geometric mean HL measured in the reference population, and in some cases were even shorter compared to the fastest eliminator in the reference population. CONCLUSION: This study suggests significantly accelerated PFAA clearance with regular phlebotomy treatment, but the small sample size and the lack of controls in this clinical intervention precludes drawing firm conclusions. Given the minimal risks of intermittent phlebotomy, this may be an effective and safe clinical intervention to diminish the body burden of PFAAs in highly exposed people.

Structural insights into the interaction of platinum-based inhibitors with the Alzheimer's disease amyloid-ß peptide.

Extended X-ray absorption fine structure spectroscopy, mass spectrometry, dynamic light scattering and density functional theory are combined to derive structural models for the interaction of neurotoxicity-ablating platinum-based compounds with the amyloid-ß peptide.

Enhanced photovoltaic efficiency via light-triggered self-assembly.

Light-initiated, radical and hydrogen-bond induced self-assembly of bis-acetamido-functionalized triarylamines is demonstrated to occur in strongly dipolar "push-pull" molecules. This self-assembly process results in the formation of self-assembled nanostructures which in turn increase the efficiency of organic photovoltaic devices.

Synthesis of ß,γ-dihydroxyhomotyrosines by a tandem Petasis-asymmetric dihydroxylation approach.

Petasis reactions of substituted styrenylboronic acids and glyoxylic acid, employing tert-butylsulfinamide as the 'amine' component, proceed with high stereoselectivity to produce ß,γ-dehydrohomoarylalanine derivatives. Subsequent asymmetric dihydroxylation under neutral conditions gives the corresponding protected ß,γ-dihydroxyhomoarylalanines with up to 15:1 dr. The method has been exploited in the efficient, stereoselective synthesis of protected ß,γ-dihydroxyhomotyrosine, a component of the antifungal cyclic peptide echinocandin B.