Anesthesia is produced by a depression of central nervous system function, however, the sites and mechanisms of action underlying this depression remain poorly defined. The present study compared and contrasted effects produced by five general anesthetics on synaptic circuitry in the CA1 region of hippocampal slices.At clinically relevant and equi-effective concentrations, presynaptic and postsynaptic anesthetic actions were evident at glutamate-mediated excitatory synapses and at GABA-mediated inhibitory synapses. In addition, depressant effects on membrane excitability were observed for CA1 neuron discharge in response to direct current depolarization. Combined actions at several of these sites contributed to CA1 circuit depression, but the relative degree of effect at each site was different for each anesthetic studied. For example, most of propofol's depressant effect (> 70 %) was reversed with a GABA antagonist, but only a minor portion of isoflurane's depression was reversed (< 20 %). Differences were also apparent on glutamate synapses-pentobarbital depressed transmission by > 50 %, but thiopental by only < 25 %.These results, in as much as they may be relevant to anesthesia, indicate that general anesthetics act at several discrete sites, supporting a multi-site, agent specific theory for anesthetic actions. No single effect site (e.g. GABA synapses) or mechanism of action (e.g. depressed membrane excitability) could account for all of the effects produced for any anesthetic studied. A-mediated inhibitory synapses and postsynaptic chloride channels [General anesthetics have been shown to depress neuronal responses in virtually all brain areas studied and this depression has been proposed to result from actions at GABAchannels -4, potaschannels -7, or cachannels -11, and/channels -17. The channels ,19. It ichannels -26 at cochannels -29. The All five anesthetics depressed synaptically evoked discharge, measured as a block of population spike (PS) responses recorded from CA1 neurons  reversed anesthetic-induced PS depression to varying degrees for each agent: isoflurane – 16.2 ± 7.4 %, halothane – 22.3 ± 18.4 %, pentobarbital – 56.2 ± 12.4 %, thiopental – 64.9 ± 12.9 % and propofol – 69.5 ± 14.3 %. Similar degrees of reversal were observed using the GABA-chloride channel blocker, picrotoxin . A GABAB receptor antagonist, CGP 55845A (10 μM) did not reverse PS depression for any of the anesthetics studied , less so for pentobarbital (~ 50 %), and contributed only partially to the depressant effects of the volatile anesthetics  were recorded from dendritic regions in stratum radiatum. All five anesthetics depressed EPSP responses  were observed in all CA1 neurons studied (n = 15) and were completely blocked by bicuculline  facilitation of Schaffer-collateral evoked EPSPs were studied. In the presence of either halothane or isoflurane no apparent change in EPSP rise time or decay kinetics were observed Fig. , contrasine Fig. . This inin vivo findings [Agent-specific effects were observed for paired pulse inhibitory responses (120 ms separation) recorded from CA1 neurons Fig. . Halothafindings  and is tfindings .per se.To determine whether the anesthetics could alter postsynaptic membrane excitability, effects on action potentials evoked by direct current injection into CA1 neurons were studied. Differences in effect were apparent across anesthetic agents – hardly any effect was evident for halothane and isoflurane, but the barbiturates and propofol produced a significant depression of action potential discharge Fig. . When meTwo conclusions can be drawn from these results: 1) for a given anesthetic, like halothane, multiple sites of action contributed in an additive manner to produce an overall depression of transmission through the CA1 neuronal circuitry Fig. ; 2) for Differing degrees of action (efficacy) were evident at both glutamate and GABA synapses for each anesthetic. For example, our results demonstrate that the two barbiturates studied appear to have differing degrees of effect at GABA synapses since thiopental's depressant effects were reversed ~ 65 % by a GABA antagonist, but pentobarbital's effects were only reversed by ~ 55 %. Similarly, these two barbiturates exhibited differing degrees of depression for glutamate-mediated excitatory inputs to the CA1 neurons, pentobarbital produced a 45 % depression in contrast to thiopental with only a 25 % depression. It was interesting that opposite actions were seen at presynaptic sites  and at postsynaptic sites . The MAS theory can readily account for the unique agent-specific profiles of effects observed in various experimental models, and also seen clinically – a long standing weakness of Unitary theories . Finallyin vivo support comes from studies utilizing a GABA beta 3 receptor mutant mouse model – proprofol-induced anesthesia was blocked in these mice, while volatile anesthetic effects were not [in vivo findings, our results indicate that effects on GABA synapses play a role in anesthetic actions, especially for propofol, thiopental and pentobarbital; but the results also indicate that effects on glutamate synapses and postsynaptic membrane excitability contribute to the CNS depression produced by all anesthetics. Given the multiple effects observed for anesthetic actions on the two types of synapses studied here, it is likely that effects on other neurotransmitter systems also contribute to anesthetic-induced depression of the CNS.Glutamate and GABA synapses in the hippocampus are among the best characterized synapses in the brain and appear to utilize receptor subtypes which are similar to those in neocortex, thalamus and other higher brain regions. Thus, the effects described in the present study would be expected to occur in these other brain regions as well, but it should be noted that different GABA and glutamate receptor subtype distributions are known to occur in cerebellum, spinal and some brain stem nuclei, and it remains to be determined whether anesthetics alter these synapses in a similar manner to their hippocampal counterparts. Ted Eger's group at UCSF has recently found that enhanced GABA-mediated synaptic transmission at the spinal level plays an important role for propofol-induced immobility in response to a noxious stimulus , but thiwere not . Taken t2/CO2, carbogen) artificial cerebral spinal fluid (ACSF). The ACSF had the following composition (in mM): Na 151.25; K 2.5; Ca 2.0; Mg 2.0; Cl 131.5; HCO3 26.0; SO4 2.0; H2PO4 1.25; and glucose 10. Whole brain coronal slices (450 μm) were cut using a vibratome (Campden Instruments), following careful removal of the dura and pia membranes. Hemisected brain slices were equilibrated for at least one hour at room temperature in an incubation chamber filled with ACSF and continually bubbled with carbogen. Individual slices were transferred to a recording chamber and equilibrated for an additional 10 minutes prior to electrophysiological recording. Oxygenated ACSF solution was continuously perfused through the chamber at a flow rate of 3.0 ml/min and maintained at 22 ± 1°C. The present studies were carried out at room temperature because synaptic responses recorded from cooler brain slices exhibit considerably better baseline stability and the tissue remains viable for many more hours in vitro compared to slices maintained at physiological temperatures. Room temperature also facilitates the use of submerged preparations (oxygen solubility and delivery to slices is increased), which allows the use of 60× optics to visualize single neurons for the patch clamp recordings used in some experiments. Previous studies comparing both volatile and intravenous anesthetic effects at physiological and cooler temperatures in brain slices found that there were no apparent differences in effects [vs. 35°C and our methods for measuring and compensating for changed aqueous solubility, as well as the remarkably similar physiological responses recorded from brain slices at these two temperatures [Male Sprague-Dawley rats were anesthetized with ether (22 vol % in air) and the brain was rapidly removed and placed in ice cold (5°C) and pregassed  were placed in stratum pyramidale to record stimulus-evoked population spike field potentials, or in stratum radiatum to record field EPSPs. Single stimulus pulses  were delivered via constant current isolation units  from a Grass S8800 two channel stimulator; at stimulus rates of 0.05 Hz. Field potential signals were amplified (× 1000), filtered , conditioned (DC offset), and digitally stored for later analysis .Whole cell patch-clamp recordings were made using thin-walled borosilicate capillaries (1.5 mm O.D.) pulled in two stages on a Narishige PP83 pipette puller. Patch electrodes were filled with the following intracellular solution (in mM): potassium gluconate or CsCl2 – 100, EGTA – 10, MgCl2 – 5, HEPES free acid – 40, ATP disodium salt – 0.3, and GTP sodium salt -0.3. The electrode solution also contained the local anesthetic QX 314 (1.0 mM) in some experiments, to prevent action potential discharge that would contaminate recordings of IPSCs. Electrode solutions were filtered and pH adjusted to 7.2 using KOH or CsOH and had a final osmolarity of 260 to 270 mOSM. Patch electrodes with a DC resistance of 4 to 5 MOhm were used. Recordings were made using an Axoclamp 2A preamplifier (Axon Instruments) in single electrode voltage clamp mode with > 80 % series resistance compensation and > 5 GOhm seals. Patch-clamp current signals were filtered , amplified (× 100) and digitized (10 KHz) for storage and analysis. Frequency and amplitudes of IPSCs were analyzed using Data Wave Technologies and Strathclyde Electrophysiological software in a continuous data recording configuration.The intravenous anesthetics  were made fresh for each experiment, solubilized using 0.5% dimethyl sulfoxide (DMSO) and sonicated immediately prior to test administration in stock solutions and serially diluted into ACSF to achieve the final concentrations for testing. Volatile anesthetics  were applied in the perfusate at equilibrated concentrations, delivered from calibrated vaporizers and bubbled into the perfusate for at least 10 min prior to switching from control ACSF, to ensure steady-state concentrations were achieved. The concentration of volatile anesthetics in the gas phase were continually measured using a Puritan-Bennett anesthetic monitor. Only a single concentration of a given anesthetic was tested on each brain slice.Data are expressed as the mean ± standard deviation and statistical analysis (ANOVA with post Tukey test) was performed using Instat from GraphPad Software. For drug effects on paired pulse inhibition, the percent change was first calculated as: Drug 1st/Control 1st = (0.5 × 100) - 100 % = 50 % depression; and Drug 2nd/Control 2nd = (X × 100) - 100 % = X % depression. Then the percent increase in paired pulse inhibition was = (X / 50) × 100 %. This approach has the advantage of normalizing paired responses with respect to varying degrees of population spike facilitation observed during control recordings, i.e. differing degrees of EPSP facilitation on a background of differing degrees of inhibition from preparation to preparation.SP and AMH contributed equally to this study by conducting most of the experimental work and they also contributed to data analysis and helped with figure preparation. MBM conceived of the study and contributed to experimental design, data analysis and interpretation of results; as well as wrote and prepared the manuscript.


Reinforcement - a process that helps prevent interbreeding between hybridising populations - is an important and little understood mechanism driving the completion of speciation Reinforcement, like sympatric speciation see , has chaSympatry—Area(s) of overlap in the ranges of populations, enabling potential interbreeding.Allopatry—Area(s) of population ranges that do not overlap with one another, preventing interbreeding.Sympatric speciation—Speciation that occurs within a range of sympatry.Reinforcement—The evolution of mechanisms that prevent interbreeding between newly interacting incipient species, as a result of selection against hybrids (narrow definition) or interspecific matings (broad definition) (See ion) See .Conspecific sperm precedence—Disproportional fertilization of a female by sperm of a conspecific male, when that female has mated with both conspecific and heterospecific males.Much of the appeal of both reinforcement and sympatric speciation lies in the way they unite micro- and macroevolution. Reinforcement, a concept popularized by Reinforcement provides a pathway toward the completion of the speciation process. Imagine that two divergent populations  come into contact after a period of allopatry . If the If the isolating mechanisms between these populations are only partially complete, extensive hybridization may occur. This can result in fusion back into a single population, or in the swamping of one population's gene pool by the genes of the other (extinction). But there is another possibility, one that can cause the speciation between the two populations to proceed. Remember that if the populations have been separated for long enough, it is likely that hybrids between them will have relatively low fitness. Individuals who mate with members of the opposing population will therefore produce offspring of poor quality, and hence have lower fitness than individuals that mate within their own population. This favors the evolution (or further divergence) of characteristics that cause mating within, rather than between, populations . SpeciatThis process, the evolution of premating isolation after secondary contact due to selection against hybrids, is reinforcement sensu Despite the substantial progress in our understanding of reinforcement that has been achieved over the last few decades, many questions remain about the process. These questions lend themselves to exploration by a broad variety of disciplines , approaches  and taxonomic systems.Doubtless, the most important unanswered question about reinforcement is how often it occurs. It is very difficult to prove that reinforcement is occurring, or has occurred, between two species. Reinforcement occasionally leaves a signature, called reproductive character displacement, in which mating characteristics have diverged between populations in areas of sympatry but not areas of allopatry  the rel. In sympDrosophila pseudoobscura and D. persimilis (Several isolated examples of reinforcement between specific pairs of species have been demonstrated, fairly conclusively, in a variety of taxa including rsimilis , flycatcrsimilis , sticklersimilis , spadeforsimilis , and walrsimilis  (see alsrsimilis . These sDrosophila (While examples such as these provide essential information about reinforcement, their slow rate of compilation and biased reporting do not provide efficient ways to assess how often reinforcement occurs in general. Comparative approaches, which examine patterns across a broader taxonomic group, can also provide support for reinforcement without these detailed mechanistic analyses (review in osophila . By compAnother area where further research is essential is the determination of which biological factors promote reinforcement, as opposed to population fusion. Theoretical studies, using mathematical models and computer simulations, are proving useful in pinpointing the effects of many factors such as migration rates and patterns, the type of selection against interspecific mating, and the genetic basis of premating isolation (reviews in In recent years, exciting developments have started to take place in the analysis of the genetics of reinforcement (reviewed in PLoS Biology illustrates the extent of the insights that can be made with this approach. Using high-resolution genetic mapping the authors have identified the locations of genes that cause increased discrimination against Drosophila persimilis males by D. pseudoobscura females, due to reinforcement in sympatry. Surprisingly, these genes map to very different areas of the chromosomes than do genes that cause a basal level of mating discrimination between the species in allopatry. Among other insights, the position of these genes suggests that the reinforced discrimination is based on odor, not on the mechanism used in allopatry, male song. This leads to the novel conclusion that reinforcement is not just increasing the strength of an already existing mechanism of species discrimination, but is occurring through the development of a new discrimination system. These kinds of developments can also motivate more realistic theoretical models of the reinforcement process.Genetic analysis is also allowing a new understanding of the mechanisms by which reinforcement might be taking place in specific cases. Work by What if, when our assessment of the frequency of reinforcement is improved, it turns out to have been a rare occurrence in the generation of current biological diversity? The study of reinforcement is broad and varied enough that many of our findings about the process would still have wide-reaching implications.First, recall the claim, at the start of this article, that studying reinforcement reveals much about the role of microevolution in the macroevolutionary process of speciation. Knowledge gained about this relationship is not only directly applicable to the very similar process of sympatric speciation, but can also tell us a great deal about speciation caused by ecological adaptation and sexual selection, which are critical components of reinforcement in many systems e.g., . StudiesSecond, analysis of reinforcement clarifies the interactions between levels of reproductive isolation that occur at different stages in the life cycle. Reinforcement, broadly defined, can be driven by isolation at the postzygotic level or by incompatibilities that occur between mating and zygote production (postmating-prezygotic incompatibilities; Finally, regardless of whether reinforcement has been a common pathway in speciation, its relevance may be increasing. Reinforcement is a possible outcome anytime species that are capable of hybridization come into contact. Human activity is increasing the incidence of secondary contact by altering habitat and introducing invasive species. This contact often results in hybridization (reviews in


Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake. Using mutated dopamine transporters, the authors find that they can separate the actions of amphetamine from the normal function of the dopamine transporter The dopamine transporter (DAT) plays a critical role in the synaptic clearance of dopamine (DA) by mediating the reuptake of DA released into the presynaptic terminal . It ther32P incorporation into DAT in response to PKC activation  , 2003. Tn = 5) in FLAG-HA-DAT relative to FLAG-DAT. Surprisingly, the DAT-mediated whole-cell currents gated by AMPH, which have been shown to be uncoupled from the transport process (In a cell suspension (or in a population of adherent cells), it is difficult to assess the potential effects on efflux of a change in ionic gradients or membrane potential because the membrane potential and ionic gradients change freely depending on the stimuli. Indeed, AMPH has been shown to induce depolarization through a DAT-mediated uncoupled chloride conductance that can be gated by substrates such as AMPH . Therefo process , were aln = 5).This reduction in current and efflux resulted from the N-terminal deletion and not from the presence of the HA epitope, since a FLAG-tagged construct lacking the first 22 amino acids of DAT (FLAG-del22-DAT) but without any other added sequence showed a reduction in current and efflux similar to that of FLAG-HA-DAT. max for uptake of the substrate tyramine by FLAG-del22-DAT was 146% that by FLAG-DAT  or cocaine  was significantly different in FLAG-DAT and FLAG-del22-DAT. Cell-surface biotinylation studies revealed that the increased Vmax in FLAG-del22-DAT was accounted for by an increased number of DAT molecules at the cell surface  that is (are) phosphorylated should lead to an effect similar to that of the truncation. Since it is not known which of the serines are phosphorylated, we simultaneously mutated all five serines to alanine in the full-length FLAG construct (FLAG-S/A-DAT). To obtain further evidence that phosphorylation of one or more of the N-terminal serines is essential for AMPH-induced DA efflux, we also created a construct in which all five of these serines were simultaneously mutated to aspartate (FLAG-S/D-DAT), in an attempt to simulate phosphorylation of the serines.m nor the Vmax for tyramine uptake was significantly different in FLAG-S/A-DAT and FLAG-S/D-DAT  or by cocaine  were not significantly different.Neither the K-DAT see . The sma-DAT see , suggestn = 6)   . Thus, the ability of AMPH to induce DAT-mediated currents and DA efflux was impaired dramatically, either by N-terminal truncation, or by substitution of the five N-terminal serines to alanine. Remarkably, substituting these five serines to aspartate to mimic phosphorylation restored the ability of AMPH to induce voltage-dependent DA efflux and to produce currents, indicating that negative charges in the DAT N-terminal region are essential for these actions of AMPH.Similarly, in FLAG-S/D-DAT cells the AMPH-induced currents and DA efflux were much greater than those generated in FLAG-S/A-DAT cells . In contrast, amperometric currents in FLAG-S/A-7D-DAT and FLAG-S/A-12D-DAT were 29.8% ± 12.6% and 45.1% ± 9.6%, respectively, of that seen in FLAG-S/D-DAT . Thus, negative charge at either position 7 or position 12 restores a substantial fraction of the efflux seen with aspartate at all five positions, and the size of the resulting efflux relative to FLAG-S/D-DAT and FLAG-DAT suggests that both of these serines may be phosphorylated in vivo (see below).To explore which serine or serines are critical to the effect on efflux, we created five additional mutants in the FLAG-S/A-DAT background in which we mutated each of the five positions, one at time, to aspartate, and we created stable pools of EM4 cells expressing each of these mutants. At +100 mV the amperometric currents in FLAG-S/A-DAT, FLAG-S/A-2D-DAT, FLAG-S/A-4D-DAT, and FLAG-S/A-13D-DAT were 7.4% ± 2.6%, 8.4% ± 5.7%, 11.2% ± 3.1%, and 12.3% ± 7.0%, respectively, of that seen in FLAG-S/D-DAT  That Dmax for uptake is unaltered in the mutants, it is possible that phosphorylation might alter the ionic coupling of DAT. The ratio of whole-cell to amperometric current . (Similar results were obtained at +60 and +80 mV [data not shown].) This ratio is a microscopic property of an individual transporter that is inversely proportional to the fraction of charge carried by dopamine  gene in pCIHyg was described previously . In the max and Km values, increasing concentrations of tyramine from 0.02 to 50 μM were used. Km and Vmax values for [3H]tyramine and [3H]dopamine uptake were determined by nonlinear regression analysis using GraphPad Prism 4. IC50 values were determined using increasing concentrations of AMPH between 0.002 and 2 μM and of cocaine between 0.001 and 10 μM in competition with approximately 60 nM [3H]tyramine. Ki values were calculated from the IC50 values as described by Uptake assays with adherent EM4 cells stably expressing the appropriate DAT construct were performed as described previously . TyraminEM4 cells stably expressing the DAT constructs were incubated with cleavable sulfo-NHS-S-S-biotin  to label surface-localized transporter, and the biotinylated material was prepared and immunoblotted as described previously .2PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, and 5.6 mM glucose) and incubated at 37oC with 15 μM DA for 30 min. Following incubation, cells were washed with KRH, harvested, resuspended in 0.20 ml of KRH and superfused in a Brandel superfusion apparatus  as described by Confluent 100-mm plates of HEK-293 cells stably expressing FLAG-DAT or FLAG-HA-DAT were washed twice with KRH  entry name for the gene discussed in this paper is S6A3_HUMAN, accession number Q01959.The Swiss-Prot (


Background to the debate: In the United States, the passage of the Bayh–Dole Act in 1980 encouraged universities to license inventions for commercial development. Although this financial incentive can stimulate academic researchers to discover new drugs and devices, there is concern that the possibility of monetary reward could distort investigators' objectivity. In the US, universities are encouraged to license inventions for commercial development. Although this financial incentive can stimulate academics to discover new treatments, it might also distort the investigators' objectivity One of the principal missions of an academic health center is to advance the understanding and treatment of disease through clinical research. In this pursuit, there is a need for checks and balances. When Jesse Gelsinger, a relatively healthy young adult, died in Philadelphia during a clinical trial of a novel adenovirus-based genetic therapy for ornithine transcarbamylase deficiency, it was a tragedy Attorney Alan Milstein made the case that the investigator and institution were both blinded to these problems by their heavy financial investment in the technology, an investment worth millions of dollars At its core, the issue revolves around conflicts of interest. In clinical research, the investigator should be primarily an advocate for the patient or volunteer. The core reason to perform clinical research is to create generalizable knowledge about a therapy, patient population, or a disease process with the long-term intent of improving human health. The interests of the patient and investigator should be fully aligned. However, most physicians in clinical research have other, more personal motivations, intermixed with the desire for progress. Successful research projects can lead to publications, promotions, grant renewals, and per case clinical trial enrollment fees. Some investigators have intellectual property rights that may have very substantial financial value if the drug or device reaches the level of approval by the United States Food and Drug Administration. These investigators stand to gain personally if the clinical trial is successful, a situation that has the potential to distort the investigator's objectivity, and may lead to a less honest relationship with study volunteers.In order to ensure that investigators are honest with potential research volunteers, the system of institutional review boards (IRBs) evolved. The IRB approves the informed consent document, which should describe the clinical experiment in a clear and dispassionate way to patients and their families. IRBs are largely made up of faculty and staff from the institution, although there are also public members and nonscientists on most IRB panels. The IRB must remain autonomous and be able to hold up or stop an investigation. There is an obligation that the IRB first and foremost think about patient rights and safety.The passage of the Bayh–Dole Act in 1980 enabled universities to license inventions for commercial development In terms of performing clinical trials using new technologies in which it has a financial interest, how is a university different from a corporate sponsor? In regard to patient safety, one primary distinction rests with the IRB. The corporate sponsor will present the research protocol to an independent commercial IRB, the university to its own IRB. Yet in both cases, there are potential conflicts of interest. The university IRB members will have a conflict of interest between the investments of their employer and the rights of the research volunteers. Independent commercial IRBs depend on pleasing corporate customers for their continued existence, and there is an unstated expectation that they will both be fast and produce rulings consistent with corporate expectations .At the university level a logical and conservative solution to the problem of institutional conflicts is to require that an IRB from outside the institution become the IRB of record when such a conflict arises. This external IRB could be either an independent commercial IRB or one of another university. The key is to grant the IRB independence and the authority to provide real oversight.There are other elements of conflicts of interest that need to be considered when the institution has a commercial interest, but most have more to do with the management of personal conflicts than institutional conflicts. The institution needs to assure the presence of an independent data safety monitoring board, thorough audits of good clinical practice, and a publications committee that will ensure submission of all meaningful study results, whether positive, negative, or neutral. Anyone subjectively evaluating patient data should be as free of conflicts as possible. These steps can be formulaically required, which should allow for performance of clinical research despite the presence of institutional commercial interests.United States research universities, and especially their academic medical centers, have greatly benefited from their uniquely privileged status in our society. That status is rooted in public confidence and trust that these institutions and their faculties will be independent and impartial in fulfilling both their academic mission to create, transmit, and preserve knowledge, and their duty to the general society to serve as credible, trustworthy arbiters of knowledge. One important mark of this status has been the remarkably consistent generosity of public support for biomedical research. Another has been the noteworthy deference of the federal government to university autonomy, and the light hand with which the sponsoring agencies historically have overseen the conduct of university research.When federal interposition occurred, it typically responded to widely publicized episodes of research misconduct, sometimes intertwined with egregious financial self interests of investigators; these episodes legitimately questioned the effectiveness of institutional oversight. Nevertheless, regulations consistently focused more on defining the metes and bounds of the permissible than on prescriptive mandates, and their implementation was effected largely through the mechanism of “assurances”—commitments that institutions would faithfully safeguard the specified perimeters of acceptable conduct.Awardee institutions thus bear primary responsibility for assuring the credibility and integrity of federally sponsored research. Public confidence in the trustworthiness of these institutions is critical, and yet nowhere is it more fragile than in biomedical research involving human participants. That confidence eroded in the 1980s and 1990s because of reports of scientific misconduct and of individual and institutional financial self interests in clinical trials. Scathing reports from federal oversight agencies and angry congressional hearings questioned whether financially self-interested institutions could any longer be trusted to guard the welfare of research participants or the integrity of clinical research.In 2001, the Association of American Medical Colleges convened a task force to examine and make recommendations on individual and institutional financial self interests in clinical research. The task force began by recognizing four important trends over the past three decades. First, the nature and culture of academic biomedical research have changed, bringing the potential of commercial relevance even to the most fundamental of scientific discoveries. Second, there has been enormous growth in the extent and depth of interactions between research universities and industry, especially in biomedicine. Third, the public has become increasingly impatient that its extraordinary investments in research yield more effective disease preventions and therapies. Fourth, the involvement of academic researchers in the translation of their discoveries has been essential in bringing those discoveries to market and to the benefit of public health.But the task force, in its two reports, asserted that both individual and institutional financial conflicts of interest in clinical research could be problematic ,5. It reBoth reports rest on a common set of core principles. The most important is that institutions should regard all significant financial interests in research involving human participants as potentially problematic. Where such interests exist, there should be a rebuttable presumption that the concerned individual or institution should not conduct the research, absent compelling circumstances. Importantly, the task force, after intense debate, rejected categorical prohibitions lest they unintentionally impede the translation of research discoveries into tangible public benefits.The task force acknowledged that the issue of institutional financial self interests is extraordinarily complex and sensitive, since it touches the very core of institutional autonomy. But the fact that an institution has a financial interest per se should raise a strong presumption against its participation in the clinical testing of that product. Public accountability and scientific integrity require that all research results emanating from academic medicine be as free as possible from the taint of financial compromise. Adding human participants to the research mix should raise the barrier to the highest level and require compelling justification for any participation by a financially self-interested institution.The task force did not define “compelling,” believing that each institution should make that determination based on disinterested scrutiny of the facts and circumstances of each case. For example, there may exist in a given institution a unique capability, without which the proposed research involving human participants could not be conducted as effectively or safely, or at all. In these instances, the public and science deserve access to that capability, provided the necessary safeguards are put in place to mediate the conflicting interests. In all such instances, protection of scientific integrity and the welfare of research participants must remain the foremost priority of both investigator and institution.This narrow window avoids absolute prohibition while striving to prevent institutional participation where credible alternatives exist. Only by such stringent self-policing can we sustain the trustworthiness and credibility of biomedical research, researchers, and their institutions, while continuing vigorously to promote the translation of biomedical discovery for the public's benefit.The public has every right to expect that academic institutions are working first for the public's interest. This value is even codified in the laws granting these institutions tax-exempt status. The public also expects that new, more effective therapies will be developed swiftly as a consequence of its support for academic research.The inventor of a new technology is always more motivated to see it through to widespread use than anyone else. This motivation, which may be as simple and benign as curiosity or as easy to understand as a financial incentive, is a powerful force driving human research. This force can be disciplined and controlled by the IRB and policies on conflicts of interest. Personal investment in research is, nevertheless, an important driver of scientific progress.When society makes an unnecessarily broad assumption that nearly all research with financial implications for investigators or their institution is potentially corrupted, a brake is placed on progress. Society will be better served by establishing clear guidelines and formalizing oversight of the research process than by rigidly limiting clinical research affected by conflict of interest. As examples, clinical trials should have independent data safety monitoring boards charged to review the study design, execution, data analysis, and publication of results. The IRB system should be strengthened in its independence through the use of community members. And, to be certain that institutional conflict of interest is avoided, an IRB from outside the institution in most cases will be preferableSociety wants better treatments. The fact that an inventor has mixed motives for developing a new treatment has always been acknowledged. The need to carefully manage the experimental process in human studies has always been understood. However, when rules minimize the role of inventors at academic centers, by forcing trials of their new ideas to go to outside institutions, society loses more than it gains. The incremental gain in safety is likely to be small (particularly if oversight is well established), while the decrease in speed of development will be significant.There are many similarities between the position espoused by Ross McKinney and my position. Most saliently, we both recognize the critically important role played by academic biomedical scientists in making discoveries and in facilitating their efficient translation into beneficial products. Neither of us proposes that academic investigators, or their institutions, should be flatly prohibited from trying to foster that translation in the presence of financial self interests.But there is an important difference. McKinney's approach for dealing with institutional conflicts of interest depends critically on the engagement of external agents to monitor closely both scientific integrity and the welfare of human participants. That, in my view, would require such deep interposition of those agents into the conduct of academic research as to be not only unprecedented but unfeasible. Beyond that, the approach falls short with respect to the maintenance of institutional trustworthiness and protection of public trust.Routine clinical assessment of technologies by financially interested institutions fosters public cynicism and distrust of the motives of academic biomedical researchers. The protective mechanisms recommended by McKinney are opaque to the public and reflect a “business as usual” image that fails fully to account for the markedly changed circumstances and perceptions of academic biomedical research. Most important, the mechanisms appear to be aimed primarily at protecting institutions' financial interests.By contrast, the American Association of Medical Colleges formulation urges that any institutional involvement in clinical research involving human participants in the presence of financial conflicts must be predicated on the presence or absence within the institution of demonstrably unique capability. This approach offers a much higher and more credible standard that aims to protect not only participant well-being and scientific integrity, but also institutional trustworthiness and public trust.


In this study, we investigated the effect of genetic background on expression profiles. We analysed the transcriptome of mouse hindlimb muscle of five frequently used mouse inbred strains using spotted oligonucleotide microarrays.Through ANOVA analysis with a false discovery rate of 10%, we show that 1.4% of the analysed genes is significantly differentially expressed between these mouse strains. Differential expression of several of these genes has been confirmed by quantitative RT-PCR. The number of genes affected by genetic background is approximately ten-fold lower than the number of differentially expressed genes caused by a dystrophic genetic defect.We conclude that evaluation of the effect of background on gene expression profiles in the tissue under study is an effective and sensible approach when comparing expression patterns in animal models with heterogeneous genetic backgrounds. Genes affected by the genetic background can be excluded in subsequent analyses of the disease-related changes in expression profiles. This is often a more effective strategy than backcrossing and inbreeding to obtain isogenic backgrounds. Due to their isogenicity, inbred mouse strains demonstrate low biological variability within each strain,2. Genetet al., manuscript in preparation). We, therefore, determined gene expression levels in hindlimb muscles from five frequently used wildtype mouse inbred strains, and compared these to the differential gene expression levels in affected muscle tissue from a mouse model (mdx) for Duchenne muscular dystrophy with healthy muscle tissue. Both the number of differentially expressed genes between strains as well as the fold-change levels are lower when compared to the differences found in affected versus healthy muscle tissue.From literature -9, it apet al., 2002), which requires an input of only 1 μg total RNA. Labelled cDNA was hybridised to murine microarrays containing 7,776 65-mer oligonucleotides spotted in duplicate.Gene expression levels in hindlimb muscle tissue from five different inbred strains  were determined. Total RNA from two individuals per strain was isolated, reversed transcribed, and subsequently labelled according to a recently developed protocol  replicate numbers[Significance levels  between the five mouse inbred strains were calculated using analysis of variance. Signifie numbers. The hige numbers.Using an FDR of 10% we selected 88 out of 6144 (1.4%) expressed genes that are differentially expressed between strains  was determined to allow a direct comparison with identical statistical methods. Selection with an FDR of 10% showed an approximately ten-fold decrease in the number of differentially expressed genes (126). Absolute fold changes were calculated and subsequently a comparison of the distribution was made  on five genes to verify our microarray data. Two genes myomesin 1 and tropomodulin 1, which were 2.2-fold and 1.8-fold lower expressed in BL6 compared to the other strains on our microarrays, were also found to be lower expressed (2.0-fold and 2.2-fold respectively) in our qPCR assay Fig. . Three omdx vs. wild-type, 1298 genes with 221 genes having a fold-change >3). Therefore, the use of mice with deviating genetic background may be justified in disease-related studies. Alternatively, strain-dependent gene expression differences may be evaluated in the initial study phase of gene targeting experiments, although the effect of hybrid backgrounds is difficult to assess.This study shows that variation in overall gene expression levels between mouse inbred strains is relatively low in hindlimb muscle tissue. This is particularly evident when the number of differentially expressed genes between two mouse inbred strains  is compared to that between diseased and healthy muscle tissue  occurring in specific blocks between mouse inbred strains. The presence of cis-acting (single nucleotide) polymorphisms may be associated with regulatory variation affecting gene expression levels. It was estimated that probably a consistent amount (up to 6%) of the roughly estimated 35,000 mouse genes contain such functional regulatory variants. We inveThis study suggests an additional method for phenotyping mouse inbred strains and provides a list of genes with significant differential expression based upon false discovery rate selection. Although overall gene expression profiles are highly similar, most significant differences are determined by low gene expression levels of BL6 compared to the other strains. A large proportion of these BL6-specific genes function as structural muscle proteins . To date, however, no major differences in muscle physiology in BL6-mice have been described which can be attributed to these reduced gene expression levels.mdx) we study. As such, the genetic background will only marginally interfere with data analysis. Determination of gene expression profiles between mouse strains enables flagging a modest number of differentially expressed genes, and is an efficient and sensible approach to circumvent tedious backcrossings, necessary to obtain isogenic animals.Perfectly isogenic backgrounds are sometimes difficult to obtain. This explorative study demonstrates that the effect of genetic background on muscle expression profiles is significant but rather limited compared to other effects, e.g. the dystrophic genetic defect (mdx/J (mdx) mice from Jackson Laboratory at the age of 6 weeks. Mice were kept under standard conditions and were sacrificed by cervical dislocation when 8 weeks old. Hindlimb muscles (m. quadriceps femoris) were dissected and promptly snap-frozen in isopentane at -80°C. Total RNA was prepared by disrupting tissue using mortar and pestle and subsequent homogenisation by a rotor-stator homogenizor  in RNA-Bee (Campro Scientific) until uniformly homogenous (15–45 sec). Total RNA was isolated according to manufacturer's instructions followed by purification using RN-easy columns (Qiagen). Quality and yield was determined using Lab-on-a-chip .We obtained CBA/CaOlaHsd (CBA), BALB/cOlaHsd (BALB), C57Bl/6JOlaHsd (BL6), DBA/2OlaHsd (DBA), and C57Bl/10ScSnOlaHsd (BL10) mice from Harland Laboratories, and C57Bl/10ScSn-Dmd6 primer (5'-NH2-(CH2)6-TN6, Eurogentec) were adjusted to a volume of 21 μl with DEPC-treated H2O , heated for 10 minutes at 70°C and chilled on ice for 10 minutes. Reverse transcription mastermix , 6 μl 5x first-strand buffer (MBI Fermentas), and 1.2 μl 25x aa-dUTP / dNTP solution ) was added per reaction and incubated at room temperature for 10 minutes followed by 2 hours at 42°C. RNA was hydrolysed by addition of 10 μl 0.5 M EDTA and 10 μl 1 M NaOH and incubation at 65°C for 30 minutes followed by neutralization by addition of 10 μl 1 M HCl. Aminoallyl labelled cDNA was then purified by combining 300 μl of PB-buffer (Qiagen) to 60 μl of the neutralized sample and centrifuged through a Qiaquick column (Qiagen) at 13000 rpm for 1 minute. Two washing steps were performed by spinning 500 μl of 75% EtOH at 13000 rpm for 1 minute while discarding the flow-through. To remove ethanol-traces the columns were centrifuged for an additional minute. cDNA was recovered by eluting three times using 30 μl basic H2O  and concentrated to a volume of 6.66 μl using a speedvac. Aliquots of Cy3 and Cy5 reactive dyes  were prepared by dissolving each vial of monoreactive dye in 40 μl fresh anhydrous DMSO (Sigma) and dividing into aliquots of 2 μl followed by vaccuumdrying until dry and subsequent storage at 4°C in the presence of silica. Fluorescent dyes were coupled by adding 3.33 μl of bicarbonate buffer  to the aa-cDNA sample and dissolving the dried aliquot of reactive dye, followed by incubation at room temperature for 1 hour in the dark. To the samples 4.5 μl 4 M hydroxylamine (Sigma) was added and incubated at room temperature in the dark for 15 minutes, followed by addition of 186 μl TE-3-buffer. Hybridisation mixtures were prepared by combining a Cy3-labeled cDNA sample with a Cy5-labeled cDNA sample and 10 μl Mouse-Hybloc  followed by removing uncoupled dyes by spinning through a pre-wetted Microcon column  for 8 minutes at 13000 rpm. Hybridisation mixture was washed by spinning 500 μl TE-3-buffer through the column and discarding the flow-through. This step was repeated two times as 2 μl yeast-tRNA  and 2 μl polyA-RNA  were added during the last step. Mixture was collected by inverting the column and spinning for 1 minute at 13000 rpm. Hybridisation mixture was finalized by adding TE-3-buffer to 84 μl together with 17 μl 20x SSC and 3 μl 10% SDS followed by denaturing at 100°C for 2 minutes, renaturing at room temperature for 15 minutes and spinning at 13000 rpm for 10 minutes. Labelled target was hybridised overnight on murine oligonucleotide microarrays . Hybridisation occurred in a automatic hybridisation station  and was followed by washing with 5x 2xSSC + 0.1% SDS at 30°C, 5x 1xSSC at 30°C, 3x 0.2xSSC at 30°C, 1x 0.2xSSC at 65°C, 2x 0.2xSSC at 30°C, and subsequently scanned as described previously[Aminoallyl labelled cDNA (aa-cDNA) was prepared based on a previously described protocol. AliquotMAANOVA2.0 The Jackson Laboratory ), hierarchical t-test [Gene expression profiles from hindlimb muscle derived from 2 male animals of each strain were generated using dye-swap experiments. Subsequent duplicate spots on each array resulted in 8 replicate measurements per gene. Targets were assigned at random to the arrays, while avoiding co-hybridisation of samples from the same strain. GenePix Pro 3.0 (Axon) was used for feature extraction and quantification. Genes were considered as being expressed when the corresponding feature was not flagged by the algorithm provided by GenePix. Local background corrected spot intensities were normalized using Variance Stabilization and Normalization (VSN) in R . Array dl t-test  and the l t-test  selectio- MuLV reverse transcriptase and accompanying buffer (MBI-Fermentas) using 1 mM dNTPs. The mixture was incubated at room temperature for 10 minutes before a 2 hour incubation step at 42°C, followed by 10 minutes at 70°C. Quantitative PCR was performed using the Lightcycler (Roche). PCR mixture was prepared by combining cDNA dilution, 10 pmol forward and reverse primer, MgCl2 (4 mM) with 4x home-made LC mastermix , Taq polymerase (0.8 U/μl), 4x SYBR Green I (Molecular Probes), 4x AmpliTaq Reaction Buffer (Perkin Elmer)) to a total volume of 20 μl. Amplicons were generated during 45 cycles with annealing temperature set at 55°C. Optimal cDNA dilutions and relative concentrations were determined using a dilution series per gene. Replicate experiments (n = 4) were normalized to 1 and relative expression values were determined by calculating the ratio per gene over the average relative expression of genes, which show no differential expression on both microarray and qPCR . Significance levels were calculated with a one-sample t-test. PCR primer pairs were designed using the Primer3 search engine, available at: Primer3 Software Distribution . The screened genes and the oligonucleotide primer pairs used for each of the genes in this study correspond to the following nucleotides: myomesin1, 4761–4780 and 4865–4884 (NM_010867); tropomodulin1, 670–689 and 878–897 (NM_021883); dysferlin, 4218–4237 and 4353–4372 (AF188290); cystatinB, 3–22 and 151–170 (NM_007793); thrombospondin4, 2167–2186 and 2289–2308 (NM_011582).qPCR was performed in duplicate for each individual resulting in four measurements per strain per gene. cDNA was prepared by reverse transcription using 1 μg total RNA as template. Random hexamers (40 ng) were used to prime the transcription after heating 10 minutes at 70°C followed by chilling on ice for 10 minutes. cDNA was synthesized by RevertAid RNaseHRT carried out the tissue preparation, total RNA isolation, target preparation, hybridisations, experimental design, data extraction, data analysis, rt-PCR, and the drafting of the manuscript. PH participated in the experimental design, analysis, rt-PCR, and study coordination. ES participated in the experimental design and analysis. RM provided statistical support. EM was responsible for mouse breeding and tissue preparation. JM participated in experimental design. GO and JD coordinated the study. All authors read the final manuscript.Differentially expressed genes between mouse inbred strains are selected with a false discovery rate of 10, 15, and 20%. Selected genes are indicated with 1, genes not selected by the specified criteria are indicated with 0. The mean of the relative gene expression levels of each of the five mouse strains is shown. For each gene the GenBank accession number is shown as well as the UniGene ID, gene description and the gene ontology description. The additional file is formatted as Comma Separated Values (CSV) file, and is named Turketal2004_Additional_File.csv.Click here for file


The authors present a method to identify modules within protein-interaction networks. Phylogenetic profiles are used to determine the biological relevance of the modules. By applying a graph-based algorithm to yeast protein-interaction networks we have extracted modular structures and show that they can be validated using information from the phylogenetic conservation of the network components. We show that the module cores, the parts with the highest intramodular connectivity, are biologically relevant components of the networks. These constituents correlate only weakly with other levels of organization. We also discuss how such structures could be used for finding targets for antimicrobial drugs. There is a strong belief underpinning systems biology that between the individual molecules and an organism's phenotype there exist intermediary levels of organization . The lowGiven that we could define such modules for a particular data source, for example, protein-protein interactions, there exists the further problem of understanding how modules relate to other forms of organization. Do for example, the proteins in a given module within a protein-protein interaction network show evidence of being coexpressed? Are they regulated by the same transcription factors and do they have the same level of dispensability?Whether we can define modules in a stringent biologically relevant fashion is not just important for our understanding of the organization of biological systems. Many authors have conjectured that if modules are real they may also be more likely to contain proteins that are essential to viability. Hence, a network approach could be imagined to hone down potential drug targets such as, for instance, candidate targets for antimicrobials.Here we ask whether phylogenetic information could be used to verify putative interaction-based modules. The assumption we make is that if a set of proteins belongs to the same module and that module has some biological relevance, then such a set should be generally conserved to act as an integrated functional unit ,19. HencL, between any given node and the rest of the nodes in the graph would exhibit a similar pattern for those nodes belonging to the same module. Alternatively, modularity could also imply that proteins within a module would interact more frequently with each other than with proteins of different modules, a property characterized by high values of a generalized clustering coefficient, C .Several network-clustering algorithms have been developed recently that make use of the local and global properties of networks -11. To tWe introduce here a simple algorithm that makes use of both sources of information. The basic steps of the so-called overlap algorithm are as follows  (discarding those ones containing just a single protein). In the next step we calculated an average overlap between both modular structures. A -value with significantly high maximal overlap was then chosen.e metric . We obtaC-based and L-based modules with a -value selected as previously described, we calculated the overlap of each C-based module with all those obtained with the L-based method. An L-based method less efficiently discriminates modular structures in small-world networks [C-based technique into a unique module. The C-based method is more robust but is weak at discriminating modules when organization levels are high. Therefore we used the C-based results as a template and the L-based method as a filter in the extraction of modular structure. In the C-based modular structure we kept in each module only those components which also appeared in the corresponding L-based module with which the selected C-module had the greatest overlap. In those cases with more than one module with maximal overlap, we selected one of them at random. Although finding the optimal classification choice is a common problem of clustering analysis, this simple algorithm allows one to select a -value with a high average maximal overlap and low overlap ratios between both methods, a measure of the reliability of the obtained modules .Having obtained networks , collapsThe overlap method was applied to the yeast protein-interaction network; that is, yeast would act as an imaginary 'poorly' characterized system where we can, however, check the relevance of our findings. This was derived from two public databases  and would be, more generally, the result of high-throughput experiments. In any case, these data are probably incomplete and no doubt contain false interactions . Should -values with significantly high average maximal overlap, that is, overlap equal to or greater than 0.8, and low ratios, characterizing the reliability of the proposed modular organizations. For an analysis of the performance of the algorithm as a function of -see Additional datafile 1. Note that these results extend the presence of modularity found previously in some yeast networks [-values in the regime described above were chosen such that the average module size is around  equal to 5 to 25 proteins, the so-called meso scale of biological networks [We therefore opted to analyze networks consisting of proteins belonging to different Munich Information Center for Protein Sequences (MIPS) protein functional categories . This alnetworks ,10,24 tonetworks   are obtained from each member of the population [To verify this initial suggestion, we examined the corresponding null hypothesis, that there is no phylogenetic correlation of the proposed structures, which is based on a completely uncorrelated distribution of phylogenetic profiles with respect to the modular organization. We made use of a class of statistical methods termed multi-response permutation procedure (MRPP). MRPPs are commonly used in ecological and environmental studies to compare an pulation . In contξ =  -  . Significance was tested by randomization; that is, we randomly permute the proteins within the modules while keeping the global modular organization fixed  to 1/2. We then sum these distances for the relevant cores in each network and estimate statistical significance by randomization Figure . We findAssuming that the putative functional group of a protein can be assigned blind to genes, this method then has the potential to narrow down the possible drug targets in poorly described species. Perhaps as expected, cell-cycle, protein synthesis and transcription-related modules have the most significant tendency to amass lethal genes. Could we apply the knowledge of validated network structures in a therapeutical context, for instance to identify targets for antimicrobials? In principle, identifying candidate proteins as antimicrobial targets is straightforward: the protein needs to be in the microbe and not the host and to be essential to the microbe. To this end, we calculated the probability of finding lethal genes in the set of proteins without human homolog belonging to the significant cores. We compared this with the probability of finding lethal genes in those yeast proteins not found in humans which are part of the full network. While the data on which genes are essential is questionable, owing to condition-dependent lethality , the ratWe have shown that by combining protein-protein data and phylogenetic information it is possible to systematically describe biologically relevant modules in protein networks which partially correlate with other types of organization. The analysis also suggests, however, that not all core modules within the functional network are equally vital for the organism's survival. This may just reflect condition-dependent lethality . Indeed,We used two databases as of July 2003: MIPS , contribL-based clustering, where L is referred to the shortest path length between two nodes in the network. From the interaction network, a matrix of distances is computed and transformed into an 'association' matrix by taking 1/L2 [C-based clustering, where C is a generalized local connectivity coefficient measuring common interactors of any two proteins in the interaction graph [Network clustering can be based on a global property, that is, ing 1/L2 . A seconon graph ,9,11 givAdj(i) the adjacency matrix, that is, the set of proteins interacting with protein i. Local properties tend to be more robust [Here |...| denotes the size of the set, ∩ the intersection and e robust .iM, jM, we considered the following overlap [Given two different modules,  overlap :) is given by:with |...| denoting the size of the set and ∩ the intersection. The average overlap used to determine the number of branches present in the clustering tree , the ratio between the number of interactions found among the m proteins connected to a given one, say j, and the maximal potential number of such interactions, which equals m(m - 1)/2 for a undirected graph. We obtained high values of such clustering coefficient and small characteristic path length for all cases, reflecting the small-worldness of the networks. To assess the statistical significance of these values, we generated 100 randomly rewired graphs for each functional network with the algorithm described in [P = 0.01), that is, , and L ≥ random L(we obtained P < 0.05 for L in the case of the energy network).To characterize the small-world property of the networks, we first calculated the clustering coefficient, ribed in . All casE-values bigger than a given threshold, thE, were considered absent [E and the absent ones receive a score of -1/logthE. For the binary case, profiles take the value 1 or 0 when the E-values are below or above the threshold, respectively. Finally, note that E-values were corrected to account for the different database sizes. Results in the main text are for the case of binary phylogenetic profiles and a threshold value of th E= 1e-6.We calculated binary and continuous phylogenetic profiles  for diffd absent . A partiNon-parametric randomization methods, such as MRPP, have several advantages compared to more well-known parametric procedures. In particular, if the assumption of normally distributed populations is not reasonable, the datasets have multiple measurements and if multivariate comparisons are desired .i, and j, we considered the following matching coefficient as a simple similarity measure: Sij = (x + w)/(x + y + z + w), where x is the number of homologs present in both phylogenetic profiles, y is the number present in profile i only and z is the number present in profile j only. Finally, w is the number of absent homologs in both profiles.Given two binary phylogenetic profiles corresponding to proteins Within similaritym cis the ratio between the number of components of module m, mn, and the number of components of all modules, MN, that is, m c= mn/MN, m Wis the mean of similarities between proteins belonging to module m, and M is the total number of modules.Here, Between similarity:m,s cis the ratio between the product of the number of components of modules m and s, m-nsn, and the total number of components squared, N2M, that is m,s c= mnsn/N2M. m,s Wis the mean of similarities between proteins of modules m and s, and M is the total number of modules. Results for all discussed tests were robust to the use of Euclidean distances with continuous profiles instead of similarities with binary profiles, as it is argued in the main text.Here, P-values for a set of k individual tests that are being performed and then rank them from smallest to largest. While Bonferroni would compare all null hypothesis to the same value α, the Holm test compares the smallest to α/k and, in case of rejection of the null case, to decreasing values α/(k - 1),... until failing to reject the null.The Holm test  is a metm W-  (see above) and determined the unadjusted P-value for each module by means of a permutation test with 10,000 randomizations. Suppose that we have M modules. We assemble an ordered vector of size M whose components are the uncorrected P-values in increasing order, that is, P1 is the smallest uncorrected P-value and PM is the largest. To adjust a particular vector component i Pwe multiply this component by Ai = (M - i + 1), thus generating a vector P for adjusted P-values. The added power of the Holm test can then be seen in a simple example. Imagine the case of three modules, that is, M = 3. The uncorrected P-values of the corresponding MRPP tests are: ν P= . A Bonferroni procedure for multiple testing would consider only the first test as significant according to a 0.05 significancy threshold. However, the adjusted P-values obtained with the Holm test would imply that all tests are significant, that is,  = ν P×  = .To perform the MRPP Holm test, we computed the branch length, that is, To obtain the core component of the modules, we selected for each module those components with more than two interactions, for the case of a module whose component with maximal number of interactions (MNI) is less than ten, or those components with more than four interactions for the case of a module whose component with MNI is equal to or greater than 10. Slight modifications to these rules produced similar results.ξm≥ 0.1, we calculated the mean of pairwise Euclidean distances between expression vectors of proteins belonging to a given module core. In the case of the 5' motifs, the statistic measures the number of regulatory motifs common to at least more than half of the core size. Finally, for each significant core, we simply measured the number of components that are lethal. The overall statistic for all cases is the sum of each corresponding measure in each core weighted by the ratio of the core size vs network size. P values are obtained with 10,000 randomizations.For each of the significant module cores, Additional data file A discussion on the network clustering algorithm, the list of species and lineages for the phylogenetic profiles, and a list of phylogenetically distinct module core components and their biological characterizationClick here for additional data file


Remarkably, a number of these proteins were previously or are now shown to be redox regulated.Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle . Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H A differential thiol-trapping technique combined with two- dimensional gel analysis has been developed and used to visualize thiol-disulfide exchange reactions, which act as switches in redox-regulated proteins Cysteine is one of the most rarely used amino acids in the proteins of most organisms studied so far . TherefoProteins in the extracellular space and oxidizing cell compartments  often rely on disulfide bonds to support their correct folding and maintain their structural stability . CytosolThe very same properties that make cysteine the perfect amino acid for redox reactions, metal coordination and thiol-disulfide interchanges, also make cysteines extremely vulnerable to oxidation by reactive oxygen species (ROS). ROS arise transiently during normal metabolism as toxic byproducts of respiration and have been shown to accumulate under conditions of oxidative stress. Over the past few years, an increasing number of thiol-containing proteins has been identified that use ROS as a mediator to quickly regulate their protein activity . This neEscherichia coli  to rapidly quench thiol-disulfide exchange reactions. All accessible thiol groups were then alkylated with cold, unlabeled IAM under denaturating conditions. In a next step, all reversible thiol modifications that developed during normal growth or oxidative stress treatment  were reduced with DTT, and the newly accessible thiol groups were modified with 14C-labeled IAM. Therefore, radioactivity was specifically incorporated into proteins that originally contained thiol modifications. High ratios of 14C activity/protein are predicted for proteins with thiol modifications while low ratios of 14C activity/protein are predicted for proteins whose thiol groups are not significantly modified in vivo  with accessible cysteine residues in proteins . Wild-ty in vivo .14C radioactivity, which correlates to the degree of thiol modification in the individual spots, was determined by exposing the dried gels to phosphor screens. Then, the 14C activity/protein ratio was visualized and quantified.The differentially trapped protein extract was then separated by 2D gel electrophoresis. Importantly, due to this specific trapping technique, all accessible thiol groups of each protein were carbamidomethylated to an extent that is independent of the original thiol-disulfide status of the protein. This ensures their identical migration behavior on 2D gels. The 2D gels were stained with colloidal Coomassie blue to get a measure of the total protein content. The E. coli. E. coli strains were grown in minimal medium to mid-logarithmic phase, and the cells were harvested. The cysteines were thiol trapped using our differential thiol-trapping technique and separated on 2D gels. To analyze the extent of thiol modification and the distribution of thiol-modified proteins in an unbiased way, we focused first on the 100 most abundant proteins on our colloidal Coomassie blue–stained 2D gels. We set the total spot intensity of all 100 protein spots on the colloidal blue gel to 100% and determined the relative spot intensity for each protein. We then quantified the 100 corresponding 14C activity spots on the phosphor image, set their combined total spot intensities to 100%, and determined again the relative spot intensity for each protein. Finally, we determined the 14C activity/protein ratios for each of the 100 proteins. Low 14C activity/protein ratios were predicted for non-thiol-modified proteins presumably present in the reducing environment of the cytoplasm, while high ratios of 14C activity/protein were predicted for proteins with cellular thiol modifications such as those present in the oxidizing milieu of the E. coli periplasm. As shown in 14C activity/protein ratio below 2.0, while nine proteins showed a higher than 2.0-fold ratio.In order to test our method, we analyzed the steady-state thiol-disulfide status of cellular proteins in wild-type 14C activity/protein ratio greater than 2.0, six are known periplasmic proteins such as the periplasmic oligopeptide permease (OppA) and glycerol-uptake protein (UgpB ), as well as proteins associated with the outer membrane like the outer membrane porin protein A (OmpA)  A and 2B.14C activity/protein ratios: dihydrolipoyl transacetylase (AceF), lipoamide dehydrogenase (Lpd), and the stringent starvation protein (SspA)  A and 2B.n (SspA) . Detecti14C activity/protein ratio below 2.0, including the very abundant elongation factor EF-Tu (Tu elongation factor [TufB]-IF1) with three cysteine residues (14C activity/protein ratio = 1.3 ± 0.5) and isocitrate dehydrogenase with seven cysteine residues (14C activity/protein = 1.2 ± 0.5). Protein spots that showed extremely low 14C activity/protein ratios (less than 0.2) included the very abundant outer membrane porin protein E as well as the cytoplasmic proteins P-specific transport protein and trigger factor , all of which do not contain any cysteine residues , ArtJ, DsbA, and the periplasmic dipeptide binding protein (DppA) were identified as proteins with a very high degree of thiol modification (14C activity/protein ratio > 2.0)  A and 2C.ase DsbA , confirmE. coli cells using our technique as well as conventional thiol-trapping methods  in our reverse-trapped samples . A reliable direct measurement of the redox potential of the dihydrolipoic acid/lipoic acid redox couple in AceF was not possible because of the very low signal that we found for AceF in the reverse-trapping experiment. This indicated that AceF is mostly in its thiol-oxidized state. This probably reflects the fact that the prosthetic group is only accessible to Lpd in its reduced state (E. coli DHB4  SDS) supplemented with 100 mM IAM. This first alkylation procedure irreversibly modified all free thiol groups that were made accessible by the urea and SDS-denaturation of the proteins. After 10 min of incubation at 25 °C, the reaction was stopped by adding 40 μl of ice-cold 20% (w/v) TCA. After 20 min of incubation on ice, the alkylated proteins were centrifuged again, and the pellet was washed with TCA as described before. The protein pellet was then dissolved in 20 μl of 10 mM DTT in denaturing buffer to reduce all reversible thiol modifications such as disulfide bonds and sulfenic acids. After a 1-h incubation at 25 °C, 20 μl of a solution of 100 mM radioactively labeled [14C-1]-IAM in denaturing buffer was added to titrate out the DTT and to irreversibly alkylate all newly reduced cysteines. The reaction mixture was incubated for 10 min at 25 °C. The reaction was stopped by adding 40 μl of 20% (w/v) TCA. After precipitation on ice and subsequent centrifugation, the pellet was washed first with TCA and then three times with 500 μl of ice-cold ethanol , and the 2D gel electrophoresis was performed as previously described .Gels were stained using colloidal Coomassie blue stain  and scan2O2 treatment at the time points 2, 5, and 30 min, the DsbA experiments under oxygen limitation, and the time course of diamide treatment. For each of the experiments, the phosphor image with the highest overall 14C activity was chosen for spot detection. The 100 most abundant spots were chosen from the detected set of spots and the boundaries transferred to all other phosphor images and protein gel images using the Delta 2D “transfer spots” function. The absolute intensity for each of these 100 spots on the protein gels and the phosphor image was determined to quantitatively describe the amount of protein and 14C activity for each protein spot. These absolute spot intensities were then normalized over all 100 spots . This normalization scheme was changed when the thiol-disulfide status of the dsbA mutant strain was analyzed. This was based on the consideration that a large number of the most intense spots on the phosphor images are heavily thiol-modified periplasmic proteins, which are putative DsbA substrate proteins. Normalizing over those protein spots would largely affect our data analysis. We therefore decided to normalize over four of the most abundant intracellular spots, TufB isoform (IF) 1, GapA IF 1, AhpC, and GroEL, whose thiol-disulfide status was not affected by the absence or presence of DsbA. In the case of the trxA mutant strain, similar considerations led us to normalize over four of the most abundant periplasmic protein spots, OmpA IF 1, OmpA IF 2, HisJ, and ArtJ, whose thiol-disulfide status was not influenced by the lack of TrxA activity. Finally, the ratio of 14C activity/protein was calculated by dividing the normalized intensity of the protein spot on the phosphor image by the corresponding normalized intensity of the Coomassie blue–stained protein spot. For a protein to be considered significantly thiol-modified, the average of the 14C activity/protein ratio for a given protein spot had to be at least 1.5-fold above the average of the 14C activity/protein ratio of this protein under control conditions.For each of the described experiments, at least four individually trapped samples of cultures were obtained from at least two independent cell cultures. The only exceptions were the time course of Hhttp://www.proteomeconsortium.org).Thiol-trapped samples using nonradioactive IAM in both alkylation steps were separated on 2D gels and used to excise proteins of interest. These proteins were identified by Peptide Mass Fingerprinting at the Michigan Proteome Consortium (http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are 30S ribosomal subunit protein S2 (P02351), 50S ribosomal subunit protein L5 (P02389), AceF (P06959), AcnB (P36683), AdhE (P17547), AhpC (P26427), and γ-glutamyl phosphate reductase (P07004), ArtI (P30859), ArtJ (P30860), aspartate semialdehyde dehydrogenase (P00353), carbonic anhydrase (P36857), carbonic anhydrase (P36857), citrate synthase (P00891), DAHP synthetase (P00886), DppA (P23847), DsbA (P24991), GapA (P06977), glutamyl-tRNA synthetase (P04805), GroEL (P06139), GTP cyclohydrolase I (P27511), HisJ (P39182), Hsp33 (P45803), Imp (P31554), isocitrate dehydrogenase (P08200), Lpd (P00391), MetE (P25665), MetQ/YaeC (P28635), NusA (P03003), OmpA (P02934), OppA (P23843), OxyR (P11721), phenylalanyl-tRNA synthetase beta-subunit (P07395), phosphate import ATP-binding protein (P07655), phosphoribosylaminoimidazole synthetase (P08178), phosphoribosylaminoimidazole-succinocarboxamide synthetase (P21155), phosphotransferase system enzyme I (P08839), PhoU (P07656), porin protein E (P02932), P-specific transport protein (P06128), pyruvate kinase I (P14178), SdhB (P07014), SerA (P08328), serine hydroxymethyltransferase (P00477), SspA (P05838), succinyl-CoA synthetase (P07459), Tpx (P37901), trigger factor (P22257), TufB (P02990), and UgpB (P10904).The Swiss-Prot (


Hürthle cell carcinoma is a variant of follicular cell carcinoma of thyroid. It may present as a low-grade tumour or as a more aggressive type. Prognosis depends upon the age of the patient, tumour size, extent of invasion and initial nodal or distant metastasis.The case of Hürthle cell carcinoma is reported in a 79-year-old man who presented with a rapidly increasing lump on the left side of his neck, having had a right hemithyroidectomy for colloid goitre 24-years-ago. Fine needle aspiration cytology confirmed the presence of Hürthle cells, raising the possibility of a Hürthle cell neoplasm. The patient underwent staging and surgery. Histology showed Hürthle cell carcinoma and the patient underwent adjuvant therapy. The literature on Hürthle cell neoplasms is reviewed.Fine needle aspiration cytology may recognise Hürthle cell lesion but final diagnosis of carcinoma depends upon histological confirmation of vascular or capsular invasion. Staging and surgery in Hürthle cell carcinoma are similar to follicular carcinoma of thyroid with favourable outcome despite the controversy regarding the histological classification and adjuvant therapy. Elderly patients with Hürthle cell carcinoma need to be made aware of their poorer prognosis and should be offered more radical treatment. He was put on a daily dose of 100 mcg of thyroxine. This was to be followed by a second dose of 5911 MBq of radioactive iodine six months from the time of the first dose.Hürthle cell carcinomas are heterogeneous neoplasms that display a wide range of biological behaviour and accounts for less than 5% of all differentiated thyroid malignancies. The term HCC should be restricted to tumours with more than 75% of oncocytic cells . OncocytOur patient had several indicators for poor prognosis such as his advanced age, male gender, large tumour size (8 cm), extra thyroid extension and nodal metastasis. Interestingly enough the patient had no pressure symptoms despite marked deviation of larynx, trachea and oesophagus, which may be due to previous right hemithyroidectomy. In elderly patients with sudden enlargement of neck mass and pre-existing thyroid conditions such as benign thyroid nodule, goitre (as in our case), Grave's disease or differentiated thyroid carcinoma, one has to bear in mind anaplastic thyroid carcinoma (ATC). In ATC local compression symptoms such as hoarseness, strider, dyspnoea and dysphagia occur as a rule -17. In aOur patient had undergone fine needle aspiration cytology, which revealed Hürthle cells. Since the lesion was rapidly growing with mediastinal extension and nodal involvement, the patient underwent staging and left hemithyroidectomy with modified neck dissection. Histology confirmed HCC based on vascular and capsular invasion. Intraoperative frozen sections have low predictive value and are particularly not a sensitive test for diagnosing HCC therefore this was not carried out . McIvor In the management of HCC the primary mode of treatment is surgical, ranging from hemithyroidectomy to total thyroidectomy. Larger tumours (>T2) require total thyroidectomy and lymphadenectomy if lymph nodes are involved . AdjuvanSeveral reports in literature have identified contra lateral foci of carcinoma in 40–70% of cases of HCC ,19. HCC 131I remnant ablation after near-total thyroidectomy [131I uptake in cervical or lung metastases [There are several reasons favouring the use of idectomy . First, tastases ,26. Secotastases . Finallytastases  and any 131I uptake at metastatic sites in only 53% of patients over 40 years of age, compared to 90% in patients below the age of 40 [131I to ablate the thyroid remnant and to treat residual disease were independent prognostic variables that favourably influenced recurrence, distant recurrence, and cancer death rates [Radioiodine therapy has no overall effect on mortality but subgroup analysis has shown that those patients who receive radioactive iodine for adjuvant ablation of remnant thyroid tissue have lower mortality rate compared with patients who either did not receive treatment or in whom the indication was the presence of residual disease . Radioioge of 40 . Univarige of 40 . Anotherth rates . Our patet al have treated 56 patients with HCC between the years 1940 and 2000 [Stojdinovic and 2000 . Of theset al in their series (42 patients with HCC between 1957–1997) used radioiodine in patients with distant metastases; none had thyroid remnant ablation with radioactive iodine [Khafif Hürthle cell lesion can be easily picked up on FNAC but to make a diagnosis of HCC one has to demonstrate vascular or capsular invasion. Intraoperative frozen sections have low predictive value and cases with advanced age (over 50), rapid enlargement of lump and palpable nodes should be regarded with high index of suspicion for presence of HCC. HCC or other differentiated carcinomas of thyroid in the elderly patients are generally more aggressive with less favourable prognosis compared to younger patients. They should be offered total thyroidectomy and selective lymph node dissection (when lymph nodes are involved) followed by ablative radioiodine therapy, provided they can withstand the above treatment. Coexisting medical disorders should be recognized and managed effectively prior to surgery . FurtherNone declared.MRH managed the patient, searched the literature and drafted the manuscriptLI: did the histological study, and contributed to pathological aspects in the present studyDG: Managed the patient, conceptualise the present report, edited the manuscript and coordinated after reviewing the manuscript


It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model , and the lacZ model . There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.The mouse spot test, an  This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test.in vivo is important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing, in vitro data must be confirmed by testing in in vivo conditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate, N-ethyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, 4-nitroquino-line-1-oxide, N-nitrosodiethylamine, N-nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and N-propyl-N-nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances quinoline (IQ), hydrazine, mitomycin C, trichloroethylene).The results on 15 out of 20 substances ; one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems.The positive results obtained with the Big Bluein vitro gene mutation assays  and in the majority of in vivo studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice.With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in N-propyl-N-nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats [in vitro gene mutation assays revealed clearly positive results.Although no data on carcinogenicity on mice is available on  in rats  and in vnot show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems  including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele , there i® mouse assay combined) and the mouse spot test are documented in Table and mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test).The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model  are integrated on chromosome 4 of each cell of the animal [In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired . Howevere animal .in vivo test.To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig  suggesteIn contrast, in transgenic mutation assays ca. 20 animals  are recommended per species and gender -121.In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic .A comparison of both test systems is presented in Table in vivo in the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript.Results in the transgenic mouse assay versus mouse spot testClick here for file