Papers by Bruce P Brandhorst
Developmental Biology, Oct 1, 1979
The 16-cell sea urchin embryo has blastomeres of three distinct size classes: micromeres, mesomer... more The 16-cell sea urchin embryo has blastomeres of three distinct size classes: micromeres, mesomeres, and macromeres. Each class is already restricted in its developmental fate, micromeres being committed to formation of primary mesenchyme cells. The three classes of blastomeres were isolated in high purity and incubated in [%]methionine until the next cleavage. Nearly all the radioactive protein was solubilixed and subjected to two-dimensional electrophoresis according to O'Farrell. Of approximately 1006 spots resolved, there are no qualitative differences among the three blastomeres. When embryos were labeled between the first and fourth cleavages and blastomeres then isolated, no qualitative differences in protein synthesis were observed. Moreover, there are very few changes when unfertilized eggs are compared to 16-cell embryos. Thus cellular determination during embryonic development is not accompanied by qualitative changes in the distribution within the embryo of abundantly synthesized proteins, virtually all of which are coded for by sequences present in the egg.
Journal of Cell Biology, May 1, 1972
The kinetics of accumulation of radioactive adenosine in adenosine triphosphate and in RNA of nuc... more The kinetics of accumulation of radioactive adenosine in adenosine triphosphate and in RNA of nuclear, cytoplasmic, and polysomal fractions of sea urchin embryos have been analyzed. 85% of the RNA synthesized decays in the nucleus with an apparently uniform half-life of about 7 min. The remaining 15% goes to the cytoplasm, mostly entering polysomes, and decays with a quite uniform half-life of about 75 min. The nuclear RNA accounts for one-third and the cytoplasmic RNA accounts for two-thirds of the total unstable RNA which accumulates at steady state in the embryo. The size distribution of shortlabeled nuclear RNA is very similar to that of long-labeled messenger RNA, when both are extracted directly from the cells without a previous cell fractionation .
Experimental Cell Research, Nov 1, 1973
A method has been developed for very rapid attainment of constant specific radioactivity of the A... more A method has been developed for very rapid attainment of constant specific radioactivity of the ATP pool in mouse L cells. In a pilot experiment, cells are labelled with a low concentration of tritiated adenosine of high specific radioactivity, and the specific radioactivity of the intracellular ATP pool is measured at various times thereafter. The principal experiment consists of Iabelling the cells with the same low concentration of tritiated adenosine, followed by addition (2 min later) of nonradioactive adenosine, such that the spec. radioact. of the exogenous adenosine becomes equal to the spec. radioact. of the endogenous ATP, which has been predicted from the results of the pilot experiment. The method avoids the long delay in equilibration of intra-and extracellular nucleic acid precursors that ordinarily occurs. It should be widely applicable to investigations of nucleic acid metabolism in eukaryotic cells, particularly to components of low stability.
Journal of Molecular Biology, May 1, 1974
The kinetics of entry of [3H]adenosine into ATP, cellular RNA, and nuclear RNA of mouse L cells w... more The kinetics of entry of [3H]adenosine into ATP, cellular RNA, and nuclear RNA of mouse L cells were determined and analyzed. A molar accumulation curve for RNA was estimated from the specific radioactivities of RNA and ATP; this curve wss resolved graphically into stable and unstable components. The stability of the unstable component (mostly heterogeneous nuclear RNA) was estimated by applying h&-order decay analysis. Heterogeneous, nuclear RNA decays with an apparently uniform half-life of 23 minutes, considerably greater than some previous estimates. It is synthesized at an instantaneous rate of 5.4 x 10e2 pg/cell per minute and reaches a steady-state level of l-8 pg/cell in the nucleus, or 7% of the total cellular RNA. Only about 2% of the heterogeneous RNA synthesized in L cells enters polysomes as messenger RNA. The implications of these values are discussed with reference to similarly determined values for sea urchin embryos.
Oversight of Stem Cell Research in Canada: Protecting the Rights, Health, and Safety of Embryo Donors
Health law review, Mar 22, 2008
Introduction Stem cell research has come to the fore of scientific and public interest in the pas... more Introduction Stem cell research has come to the fore of scientific and public interest in the past decade, bearing the promise of providing treatments for many serious diseases and conditions. (1) Yet the pursuit of this research has raised significant issues of public policy and ethics in Canada and elsewhere. Recent policy discussion centers on developing ways of overseeing stem cell research that are consistent with the regulation of other forms of scientific research and yet take into account distinctive aspects of this research. (2) Ethical issues revolve around the derivation, study and research use of human pluripotent stem cells within the bounds of the ethical requirements of the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans (TCPS). (3) In 2002, the Canadian Institutes of Health Research (CIHR) developed guidelines to address the oversight and associated ethical issues raised by stem cell research, the Human Pluripotent Stem Cell Research Guidelines (the Guidelines). (4) It also established the Stem Cell Oversight Committee (SCOC) to ensure that all pluripotent stem cell research carried out at institutions receiving funding from the Tri-Council Agencies--and, on a voluntary basis, at other public or private granting agencies in Canada or within the private sector--is in accordance with the Guidelines. (5) The SCOC consists of a chair and a minimum of 11 additional members chosen by the CIHR Governing Council on advice from the Nominating Committee. (6) It is structured to be: a heterogeneous group of individuals with a range of backgrounds and disciplines relevant to the mandate of the Committee. Technical experts will provide the Committee access to the latest scientific and ethical information, and representatives from the general public will represent the views and values of Canadians potentially affected by the new technologies. (7) Members of the SCOC include professionals in stem cell biology and therapeutics; developmental biology or embryology; health care (in vitro fertilization [IVF] specialist); ethics; law; international stem cell research policy; and the social sciences. Persons from the voluntary health sector, IVF patients, and members of the general public who have a general interest in health research (8) and who are not advocates for any specific interest group are also included. (9) Members of the SCOC are not employees of CIHR and do not receive an honorarium or remuneration of any kind from CIHR. The SCOC was mandated to provide periodic updating and proposals for revision of the Guidelines to the CIHR Governing Council and has done so twice, once in 2005 (10) and again in 2006. (11) Important questions have recently been raised by several commentators about modifications made in the Guidelines before and after their publication. Others have questioned certain requirements of the Guidelines that they believe are too restrictive. In this article, those who were members of the SCOC from 2003 to 2006 trace certain clarifications of the Guidelines made during that period and discuss the rationale for them in light of these concerns. They also address criticisms made of the substance of the Guidelines. The thrust of this article is to exhibit that the paramount consideration underlying the development of the Guidelines has been the need to protect the rights, health, and safety of those who donate embryos for human pluripotent stem cell research. I. Major Provisions of the CIHR Guidelines for the Oversight of Stem Cell Research The Guidelines permit research to study human embryonic stem cell lines derived from human embryos created but no longer required for reproductive purposes, as well as preexisting human embryonic stem cell lines. (12) They mandate that persons for whom the embryos were created, and third parties who have donated gametes for the development of embryos, must have given free and informed consent for the research use of such spare embryos. …
Restricted expression of the Lytechinus pictus Spec1 gene homologue in reciprocal hybrid embryos with Strongylocentrotus purpuratus
Developmental Biology, Apr 1, 1991
Hybrid embryos were derived from reciprocal crosses of Strongylocentrotus purpuratus and Lytechin... more Hybrid embryos were derived from reciprocal crosses of Strongylocentrotus purpuratus and Lytechinus pictus sea urchins. The expression of proteins specific for L. pictus was restricted in these hybrid embryos, while this was not so for most proteins specific for S. purpuratus. In particular, the aboral ectoderm-specific calcium-binding protein Spec1 was expressed at normal levels in hybrid embryos, but its L. pictus homologue, LpS1, was considerably reduced. LpS1 mRNA accumulated in hybrid plutei to only 4-5% of its normal level. Transcription of the LpS1 gene was substantially reduced in hybrid embryos, as determined by a nuclear RNA run-on assay. Southern blot analysis of genomic DNA indicated that there was no detectable loss or rearrangement of LpS1 DNA in hybrid embryos. Thus, the Spec1 gene is expressed normally in hybrid embryos, but the transcription of its homologue, the LpS1 gene, is considerably restricted.
Informational Content of the Echinoderm Egg
Springer eBooks, 1985
The sea urchin egg contains a store of mRNA synthesized during oogenesis but translated only afte... more The sea urchin egg contains a store of mRNA synthesized during oogenesis but translated only after fertilization, which accounts for a large, rapid increase in the rate of synthesis of largely the same set of proteins synthesized by eggs. Starfish oocytes contain a population of stored maternal mRNA that becomes actively translated upon GVBD and codes for a set of proteins distinct from that synthesized by oocytes. The sequence complexity of RNA in echinoderm eggs is about 3.5 x 10(8) nucleotides, enough to code for about 12,000 different mRNAs averaging 3 kb in length. About 2-4% of the egg RNA functions as mRNA during early embryonic development; most of the sequences are rare, represented in a few thousand copies per egg, but some are considerably more abundant. Many of the stored RNA sequences accumulate during the period of vitellogenesis, which lasts a few weeks. The mechanisms of storage and translational activation of maternal mRNA are not well understood. Histone mRNAs are sequested in the egg pronucleus until first cleavage, but other mRNAs are widely distributed in the cytoplasm. The population of maternal RNA includes many very large molecules having interspersed repetitive sequence transcripts colinear with single-copy sequences. The structural features of much of the cytoplasmic maternal RNA is thus reminiscent of incompletely processed nuclear precursors of mRNA. The functional role of these strange molecules is not understood, but many interesting possibilities have been considered. For instance, they may be segregated into different cell lineages during cleavage and/or they may become translationally activated by selective processing during development. Maternal mRNA appears to be underloaded with ribosomes when translated, possibly because the coding sequences are short relative to the size of the mRNA. Most abundant and many rare mRNA sequences persist during embryonic development. The rare sequence molecules are replaced by newly synthesized RNA, but some abundant maternal transcripts appear to persist throughout embryonic development. Most of the proteins present in the egg do not change significantly in mass during development, but a few decline or accumulate substantially. Together, these observations indicate that much of the information for embryogenesis is stored in the egg, although substantial changes in gene expression occur during development.
Wilhelm Roux's archives of developmental biology, 1979
Strongylocentrotus purpuratus embryos were reared in 0.025 M LiC1, which causes commitment to veg... more Strongylocentrotus purpuratus embryos were reared in 0.025 M LiC1, which causes commitment to vegetalized development within 5 h after treatment begun at fertilization. Treated and control embryos were labelled with 3SS-methionine for 3 h intervals from 2-14 h, solubilized, and subjected to 2-dimensional polyacrylamide gel electrophoresis. Comparison of autoradiographs of the gels, in which over 400 proteins can be detected, indicate that while LiC1 treatment causes a short delay in the initiation or cessation of synthesis of a few proteins, no qualitative or major quantitative differences can be detected between control and treated embryos. Normal gastrulae and vegetalized exogastrulae labelled 38 h after fertilization have several differences in patterns of protein synthesis. We conclude that the early determinative events involved in vegetalization are not reflected in detectable differences in the pattern of protein synthesis.
Genes & Development, 1990
Expression of the Spec3 gene of Strongylocentrotus purpuratus is associated with ectodermal cilio... more Expression of the Spec3 gene of Strongylocentrotus purpuratus is associated with ectodermal ciliogenesis. An antiserum was raised against the amino terminus of the deduced Spec3 amino acid sequence and used for immunofluorescent staining. Cilia and an apical structure at the base of the stained cilium of each ectodermal cell stained intensely in gastrula and later stage embryos. Microtubule-depolymerizing agents dispersed the concentrated spot of apical staining, suggesting a localization of Spec3 antigen to the Golgi complex. Immunogold electron microscopy confirmed the localization of Spec3 antigen on cilia and in the Golgi complex. Spec3 antigen showed a diffuse punctate staining pattern in the ectodermal cytoplasm of hatching blastula when Spec3 transcripts are most prevalent, suggesting that after synthesis, Spec3 is sequestered in the Golgi complex before appearing on cilia. Whereas the predicted M, of the Spec3 protein is 21,600, immunoblotting with S. purpuratus proteins indicated that a Spec3 antigen was concentrated in cilia and migrated as an SDS-resistant aggregate of Mr-350,000. Spec3 is also concentrated in cilia of Lytechinus pictus but the protein migrated with an M,-23,000 in this species. The S. purpuratus Spec3 antigen remains associated with the ciliary axoneme after extraction of membrane proteins.
Development, 1988
When pluteus embryos of Lytechinus pictus were treated with colcemid, the incorporation of [ 35 S... more When pluteus embryos of Lytechinus pictus were treated with colcemid, the incorporation of [ 35 S]methionine into tubulin declined by 5-to 15-fold within 4 h. This was mostly accounted for by a rapid decline in the concentration of a-and /2-tubulin mRNA in the cytoplasm. Treatment with other microtubule depolymerizing agents (colchicine, nocodazole, low concentrations of vinblastine) had similar effects. Treatment of embryos with the microtubule-stabilizing agent, taxol, or high concentrations of vinblastine resulted in increased synthesis of tubulin. The concentration of tubulin mRNA increases during development and becomes increasingly sensitive to colcemid and decreasingly sensitive to taxol. The transcriptions! activity of tubulin genes, estimated by an RNA run-on assay in isolated nuclei, was not altered after colcemid treatment. On the other hand, the rate of decay of tubulin mRNA in prism embryos treated with actino-mycin D was increased several fold by colcemid treatment, while taxol treatment led to an increased half-life of tubulin mRNA. These observations suggest that tubulin synthesis is autogenously regulated at the level of mRNA stability by the level of unpolymerized tubulin. The increasing polymerization of microtubules and declining level of unpolymerized tubulin during embryogenesis would result in a stabilization of tubulin mRNA accounting for the increasing concentration of tubulin mRNA and rate of tubulin synthesis, as well as the increasing sensitivity of tubulin synthesis to microtubule-depolymerizing agents. Evidence is also presented for a rapid influence of the level of unpolymerized tubulin on the efficiency of translation of tubulin mRNA.
Molecular Patterning along the Sea Urchin Animal-Vegetal Axis
International review of cytology, 2002
The molecular regulatory mechanisms underlying primary axis formation during sea urchin developme... more The molecular regulatory mechanisms underlying primary axis formation during sea urchin development have recently been identified. Two opposing maternally inherited systems, one animalizing and one vegetalizing, set up the animal-vegetal (A-V) axis. The vegetal system relies in part on the Wnt-beta-catenin-Tcf/Lef signaling pathway and the animal system is based on a cohort of animalizing transcription factors that includes members of the Ets and Sox classes. The two systems autonomously define three zones of cell-type specification along the A-V axis. The vegetalmost zone gives rise to the skeletogenic mesenchyme lineage; the animalmost zone gives rise to ectoderm; and the zone in which the two systems overlap generates endoderm, secondary mesenchyme, and ectoderm. Patterning along the A-V also depends on cellular interactions involving Wnt, Notch, and BMP signaling. We discuss how these systems impact the formation of the second axis, the oral-aboral axis; how they connect to later developmental events; and how they lead to cell-type-specific gene expression via cis-regulatory networks associated with transcriptional control regions. We also discuss how these systems may confer on the embryo its spectacular regulatory capacity to replace missing parts.
Developmental Biology, Mar 1, 1983
We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Str~lg... more We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Str~lgylocentrotus ~ur~rut~s by two-dimensional gel eiectrophoresis. There was an increase in the number of proteins detectably synthesized during development, as well as significant changes in relative rates of synthesis invoking approximately 20% of the nearly 900 newly synthesized polypeptides. The majority of these changes were increases rather than decreases in synthesis; about half were of at least lo-fold, while a few were of more than loo-fold. Very few changes were detected upon fertilization and during the first several hours of development, while about 60% of the changes detected occurred between the hatching and the beginning of invagination. An analysis of proteins detected by silver staining indicated that most remained nearly constant in mass during embryonic development, but several increased or declined substantially. Many proteins present in eggs were not detectably synthesized in either eggs or embryos.
Expression of Maternal and Embryonic Genes during Sea Urchin Development
Springer eBooks, 1984
We have investigated the patterns of protein synthesis during embryonic development using two-dim... more We have investigated the patterns of protein synthesis during embryonic development using two-dimensional electrophoresis. The recruitment of a large amount of new maternal mRNA into polysomes following fertilization is accompanied by very few changes in the relative rates of synthesis of about 1000 polypeptides detected. Between the time of hatching and invagination, many polypeptides become undetectible and many others appear. A variety of other developmental changes occur in this period as well, indicating that a key transition has occurred from early development, dominated by expression of the maternal genome to late development, which requires participation of the embryonic genome. About 20% of the newly synthesized polypeptides, mostly minor ones, undergo changes in relative rates of synthesis during development, and far fewer change detectibly in mass. A variety of patterns of metabolism are observed for different polypeptides. Cloned cDNAs have been identified which correspond to mRNAs coding for some of these developmentally regulated proteins. Some of the newly synthesized polypeptides are enriched in one of the three primary tissue layers, and these increase in relative rates of synthesis during development. A comparison of polypeptides enriched in ectoderm or endoderm demonstrates little evolutionary conservation among three genera. Few prevalent paternal mRNAs are detectible in comparable abundance in interspecies hybrid embryos even at the mature pluteus stage. For some genes this may be because the stored maternal RNA persists throughout embryonic development without being replenished by new synthesis. We have identified several cloned DNAs complementary to transcripts which normally accumulate extensively during embryogenesis of the paternal species but are barely detectible in the hybrid embryos. The possible defects in expression of these paternal genes in foreign cytoplasm are discussed.
Assignment of sockeye salmon (Oncorhynchus nerka) to spawning sites using DNA markers
Marine Biotechnology, Jun 4, 2005
Randomly amplified polymorphic DNA (RAPD) markers were used to assign individual adult sockeye sa... more Randomly amplified polymorphic DNA (RAPD) markers were used to assign individual adult sockeye salmon to their spawning sites using a genotype assignment test. Six primers were selected for use by screening bulked DNA samples for markers missing in fish from one or more of 5 sites in British Columbia or Alaska. Of 73 markers scored, 54 showed variation between or within sites among the sampled fish. Thirty-seven of the variable markers were not detected in any fish from one or more sites; 18 variable markers were detected in all fish from one or more other sites. Thus 25% of markers scored were found in all fish of some sites and in no fish of some other sites. An assignment test placed all 70 fish tested into their correct populations. Principal coordinate analysis of genetic variation produced clusters of fish corresponding to each sampling site. No sex-specific RAPD markers were detected among more than 1300 screened.
Mechanisms of Development, 2011
Glycosaminoglycans (GAGs) are a heavily sulfated component of the extracellular matrix (ECM) impl... more Glycosaminoglycans (GAGs) are a heavily sulfated component of the extracellular matrix (ECM) implicated in a variety of cell signaling events involved in patterning of embryos. Embryos of the sea urchin Strongylocentrotus purpuratus were exposed to several inhibitors that disrupt GAG function during development. Treatment with chlorate, a general inhibitor of sulfation that leads to undersulfated GAGs, reduced sulfation of the urchin blastocoelar ECM. It also prevented correct specification of the oral-aboral axis and mouth formation, resulting in a radialized phenotype characterized by the lack of an oral field, incomplete gastrulation and formation of multiple skeletal spicule rudiments. Oral markers were initially expressed in most of the prospective ectoderm of chlorate-treated early blastulae, but then declined as aboral markers became expressed throughout most of the ectoderm. Nodal expression in the presumptive oral field is necessary and sufficient to specify the oral-aboral axis in urchins. Several lines of evidence suggest a deregulation of Nodal signaling is involved in the radialization caused by chlorate: (1) Radial embryos resemble those in which Nodal expression was knocked down. (2) Chlorate disrupted localized nodal expression in oral ectoderm, even when applied after the oral-aboral axis is specified and expression of other oral markers is resistant to treatment. (3) Inhibition with SB-431542 of ALK-4/5/7 receptors that mediate Nodal signaling causes defects in ectodermal patterning similar to those caused by chlorate. (4) Intriguingly, treatment of embryos with a sub-threshold dose of SB-431542 rescued the radialization caused by low concentrations of chlorate. Our results indicate important roles for sulfated GAGs in Nodal signaling and oral-aboral axial patterning, and in the cellular processes necessary for archenteron extension and mouth formation during gastrulation. We propose that interaction of the Nodal ligand with sulfated GAGs limits its diffusion, and is required to specify an oral field in the urchin embryo and organize the oral-aboral axis.
On nitric oxide signaling, metamorphosis, and the evolution of biphasic life cycles
Evolution & Development, Sep 1, 2003
Summary Complex life cycles are ancient and widely distributed, particularly so in the marine env... more Summary Complex life cycles are ancient and widely distributed, particularly so in the marine environment. Generally, the marine biphasic life cycle consists of pre‐reproductive stages that exist in the plankton for various periods of time before settling and transforming into a benthic reproductive stage. Pre‐reproductive stages are frequently phenotypically distinct from the reproductive stage, and the life cycle transition (metamorphosis) linking the larval and juvenile stages varies in extent of change but is usually rapid. Selection of suitable adult sites apparently involves the capacity to retain the larval state after metamorphic competence is reached. Thus two perennial and related questions arise: How are environmentally dependent rapid transitions between two differentiated functional life history stages regulated (a physiological issue) and how does biphasy arise (a developmental issue)? Two species of solitary ascidian, a sea urchin and a gastropod, share a nitric oxide (NO)‐dependent signaling pathway as a repressive regulator of metamorphosis. NO also regulates life history transitions among several simple eukaryotes. We review the unique properties of inhibitory NO signaling and propose that (a) NO is an ancient and widely used regulator of biphasic life histories, (b) the evolution of biphasy in the metazoa involved repression of juvenile development, (c) functional reasons why NO‐based signaling is well suited as an inhibitory regulator of metamorphosis after competence is reached, and (d) signaling pathways that regulate metamorphosis of extant marine animals may have participated in the evolution of larvae.
Biochemistry, Mar 1, 1971
Developmental Biology, Apr 1, 1978
The kinetics of accumulation of nuclear and cytoplasmic poly(A) have been determined in sea urchi... more The kinetics of accumulation of nuclear and cytoplasmic poly(A) have been determined in sea urchin blastulas and gastrulas, stages when essentially all mRNA is synthesized de noL)o in the nucleus. A majority of the labeled poly(A) is found in the cytoplasmic fraction after a brief pulse. The ratio of radioactive AMP to adenosine in pulse-labeled nuclear, cytoplasmic, and pol.yriboso-ma1 poly(A) is considerably less than the number average length of the labeled poly(A), indicating that there is 3'-terminal addition of adenosine to previously synthesized poly(A). The size distribution of pulse-labeled, terminally elongated poly(A) in the cytoplasm is similar to that of the largest nuclear poly(A) rather than the steady-state size distribution of cytoplasmic poly(A), which is smaller and more heterogeneous. The most likely interpretation of these results is that there is a predominant 3' terminal addition of short tracts of adenosine to poly(A) attached to nuclear RNA just before or during entrance of this RNA into the cytoplasm. In this respect, much of the 3' terminal addition may be thought of as terminal completion of poly(A) synthesis.
Developmental Biology, Jul 1, 1982
We have used two-dimensional electrophoresis to analyze the synthesis of paternal proteins in emb... more We have used two-dimensional electrophoresis to analyze the synthesis of paternal proteins in embryos of three interspecies hybrids which form healthy pluteus larvae: the reciprocal crosses of Strongylocentrotus purpwatus and Strongylocentrotus droebachiensis as well as S. purpuratus eggs fertilized with Lytechinus pictus sperm. No proteins specific to the paternal species were detectably synthesized at the two-to four-cell stage. By hatching blastula stage the synthesis of a few proteins specified by the paternal genome was detected, but these did not increase significantly during later development, and the synthesis of most distinctly paternal proteins was never detected. Radioactive cDNA probes were prepared by reversed transcription of polysome-enriched polyadenylated RNA of S. purpuratus or L. pictus gastrulae. The rate and extent of annealing of these probes to homologous sperm DNA or hybrid embryo DNA indicated that DNA coding for mRNA normally translated in embryos of the paternal species is fully retained in S. purpuratus x L. pi&us hybrid gastrulae. Hybridization of these probes to excess cytoplasmic polyadenylated RNA of hybrid embryos indicated substantial underrepresentation of paternal transcripts, particularly those which are normally prevalent. These observations may be explained if much of the mRNA translated into proteins detected on twodimensional gels is persistent maternal RNA. Alternatively, the synthesis or processing of many paternal mRNA sequen:es may be impaired in the hybrid embryos.
Translational activation of maternal mRNA encoding the heat-shock protein hsp90 during sea urchin embryogenesis
Developmental Biology, Sep 1, 1986
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Papers by Bruce P Brandhorst