Standard approaches to communicating risk to patients do not appear to be very effective, argues Alaszewski. We need a new approach that takes patients' own perceptions into account Doctors and other health professionals play a key role in communicating risk information. They are advisers to patients, especially when patients have to make fateful decisions that can irrevocably change their lives. There is a developing body of literature on the ways in which risk information can be effectively communicated ,2. HowevMuch of the discussion of risk communication is grounded in the rational model of risk communication ,4. This not used risk knowledge effectively, then the response of the professional is to consider ways in which risk communication can be improved, such as by improving its presentation or mode of communication. When patients appear to be making irrational or harmful decisions, for example, continuing to smoke, choosing not to vaccinate a child against measles, mumps, and rubella, or not complying with medication, the professional's response is to work harder to convey the risks.With the rational model, when there is evidence that patients have But the rational model contains two key flaws. One relates to the nature of risk knowledge and the second to the nature of communication . Within populations. Individual patients need information on their own personal risks. Expert assessments of risk tend to focus on the knowable and measurable components of risk, that is, the extent to which future events are the same as and predictable by the knowledge of past events. Such assessments by definition exclude uncertainty—those aspects that cannot be assessed and measured. Given the speed of social and technological change, it is not clear that the past is an effective guide to the future. As such, there is an increasing awareness of the uncertainty of risk assessment, for example, in relationship to new diseases such as HIV/AIDS or new technologies such as mobile phones or genetically modified foods.Risk knowledge cannot actually be used directly by patients to inform their decision-making. Scientific research such as in epidemiology generates knowledge about the probability of harmful events occurring within www.cochrane.org) they can find the latest evidence-based assessments of medical treatments and technologies, or via the Dr. Foster Web site (www.drfoster.co.uk) they can find the risks associated with different treatment facilities in the United Kingdom. Many patients access a variety of different sources, so they can clearly compare and evaluate the information provided by each.Within the rational model of risk communication, the emphasis is on the flow of knowledge from the knowledgeable doctor to the uninformed patient. However, communication is a two-way process, and increasingly there is awareness of the active role of patients and the public . PatientPatients do give particular credibility to sources that they know, which may include family and friends but also medical advisers with whom they have developed a relationship. They are particularly concerned about the trustworthiness of particular sources. While individuals can use their personal experience to evaluate the trustworthiness of personal sources, such as a particular relative or doctor, they often use contextual information to judge the trustworthiness of impersonal sources . For exaPatients will actively interpret risk information. If the information is timely and relevant it will tend to be accepted. Patients tend to define relevance in terms of the way they view or frame a situation, and there may be considerable differences between the ways that experts and patients view the same situation. As Zinn notes, the ways in which individuals frame and perceive risk will be highly influenced by their social situation, especially their personal biography . IndividStandard approaches to risk communication, whether targeted at groups or individuals, do not appear to be very effective. For example Ruston and Clayton have shown the ways in which women disregard information and conceptually distance themselves from the risk of coronary heart disease—this applies even to those admitted to hospital with the disease . ColemanThus, the initial stage of communication could involve identifying the key issues, that is, those that cause concern for the patient. In this phase the emphasis might be on the patient talking and the doctor listening. If there is a major difference in the ways in which patient and doctor are framing the risk issues, then there might need to be an exchange or negotiation in which both parties adjust their mutual expectations and seek a mutually acceptable definition of what the problem is. If such an exchange does not take place, and if the patient's definitions are disregarded and not acknowledged, there is the danger that the patient will passively acquiesce but treat much of the information provided by the doctor as irrelevant and disregard it.If and when there is agreement, then there is the possibility of discussing the future and the likely consequences of taking different actions . During this part of the exchange the emphasis might shift towards the doctor talking more and the patient listening more. There is a transfer of information, but it is a two-way process. The doctor should learn something about the patient's situation, including the risks that the patient is concerned about and the patient's beliefs about the nature of such risks. The patient should learn about the doctor's views of the nature of the risks that the patient is facing and the options for managing such risks.Underpinning the development of an effective relationship is the development of mutual trust. While trust usually takes time to develop, it is possible even during a short but positive exchange, in which mutual respect is shown, for a form of “swift” trust to be developed .While I have focussed on communication in face-to-face relationship, the same issues and processes can be identified in more impersonal communication, such as the provision of risk information in health promotion campaigns. In such campaigns special mechanisms need to be created for dialogue. For example, Jones has described a project that engages young drug users in Hong Kong by helping them make videos about drug use, and has shown how such techniques can be used to evaluate and improve current health promotion adverts .There are no quick technical fixes for communicating risk information. If health professionals are serious about communicating risk information so that patients and others can make informed choices, they need to recognise that communication is a two-way process, and they need to take time to access patients' accounts and perceptions. Such investment should pay off both in an improved relationship and also in improved concordance with treatments.


Of the 300 or so viruses that cause disease in humans, HIV may have the greatest adaptive advantage. Like most persistent viruses—including the herpesviruses Epstein–Barr and cytomegalovirus (CMV)—HIV employs various strategies to counteract its host's response to infection. But HIV possesses a unique ability to sustain a progressive attack on the immune system—infecting the very cells that coordinate the immune response—leaving the body susceptible even to normally harmless microorganisms. It is these so-called opportunistic infections, rather than the human immunodeficiency virus itself, that makes HIV so deadly. The specific mechanisms that engineer this ongoing systemic attack have been the subject of intense research.HIV targets white blood cells with protein surface receptors called CD4. These CD4, or helper, T-cells normally orchestrate the body's immune response by signaling killer T-cells  and other immune cells to multiply and differentiate—that is, become specially equipped to recognize a particular pathogen, or antigen. At the onset of infection, the immune system appears to respond normally, with a strong attack led by HIV-specific CD8 T-cells that initially contain the virus. But as the infection progresses, CD4 counts drop and the body's ability to renew T-cells decreases while its proportion of “antigen-experienced” CD8 T-cells increases. While the biological effect of this hyperactivity is unclear, it is apparent that patients with elevated immune activity face a poor prognosis. Investigating the interaction among immune activation, CD8 T-cell differentiation, and HIV prognosis, Victor Appay and colleagues report that a close connection between elevated immune activation and elevated levels of highly differentiated T-cells may bring further insights into how HIV exhausts the immune system.To examine the effect of elevated immune activation on T-cells, the researchers analyzed T-cells from a group of HIV-infected individuals collected at two distinct points in time: at the onset of acute infection—which is characterized by vigorous HIV replication—and after treatment, when viral replication is suppressed. To explore the connection between T-cell differentiation and clinical status, the researchers analyzed the T-cells from a group of untreated infected individuals divided into three subsets based on stage of infection: acute infection, chronic infection without progression, and chronic infection with signs of progression.During acute HIV infection, the vast majority (80%–90%) of the CD8 T-cell population was activated—not just the HIV-specific CD8 T-cells. Surprisingly, CD8 T-cells specific to the Epstein–Barr and CMV viruses showed significant activation levels during acute infection, suggesting that HIV may indirectly promote the replication of these viruses. When the researchers investigated the effects of this activation on T-cell differentiation, they found a correlation between increasing antigen concentrations and increasing CD8 T-cell activation and proliferation. And when Laura Papagno et al. analyzed the differentiation state of CD8 T-cells in individuals at different stages of infection, they found a progression in the proportion of highly differentiated CD8 T-cells associated with HIV disease progression.These results, the researchers conclude, show that chronic overactivation of the immune system during HIV infection produces the large pool of highly differentiated T-cells observed in HIV infection. T-cells go through various stages toward late differentiation, and it may be that the early-differentiated CD8 T-cells, which maintain the ability to proliferate, offer protective immunity. But highly differentiated cells, they propose, exhibit characteristics associated with “replicative senescence”—they are in effect old, worn-out cells that can no longer proliferate. Though replicative senescence is a natural process for most cells, in the context of HIV—in which infected individuals also lose the ability to replenish T-cells—it creates an aging population of T-cells that are less effective at fighting infection.


The legacy of Francis Crick is explored by two scientists who were influenced by his work He believed that scientific understanding removed the need for religious explanations of natural phenomena. From James Watson's and his early work, the structure of DNA explained the α, the origins of life. This was a starting point; from the elucidation of the structure of DNA, there was an explosion, a massive diversity of science that in part removed the need to postulate a creator or a creation myth. Francis still felt that life was no less astonishing just because it was biological and natural in origin. He had a consistent and completely rational world view without a need to invoke vitalism, or any non-material force . And in Once the structure of DNA was known, the physicist George Gamow formed the RNA Tie Club, with Francis and eighteen others including his close friends Leslie Orgel and Sydney Ten years later, the secrets of DNA transcription and translation unmasked, Francis turned to consciousness. He admitted he knew little at first, only that the structure of consciousness was as tough a problem as DNA's structure. DNA was certainly not played out, but the Ferrier Lectures in the Proceedings of the Royal Society of London by David Hubel and Torsten Wiesel were just available, tempting Francis with an almost physicist's view of neurons in action. Hubel and Wiesel wrote of functional architectures, embedded in beautiful, almost crystalline structure. The comprehension of mind invoked by a biological mechanism appeared ripe for the sort of thoughtful, theoretical science he had applied to DNA. Francis was now sixty years old and moved from Cambridge to the Salk Institute in La Jolla, California. Francis began with the brightest young minds he could find.David Marr was a young mathematician and physiologist whose doctoral thesis on a theory of mammalian brain function at Cambridge had brought him into some contact with Brenner and Francis. A professor at the Massachusetts Institute of Technology, he began working with Tomasio Poggio of the Max Plank Institute in Tübingen on a computational theory of neuroscience. Following an invitation from Francis, Poggio and Marr spent the month of April, 1979 extending their intense examination of the core problems of visual perception. They spent hours sitting at the most western end of the Salk Institute, at the cafeteria or in Francis's office, gazing into the Pacific Ocean with all its daily changes, discussing not only architecture of visual cortex and visual perception, but the ramifications of a good theory of brain function. We know of these conversations, as the probing of Marr by Francis is captured in the final chapter of Marr's now classic book “Vision” . (AlthouMarr had been diagnosed with acute leukemia in the winter of 1978 . The oneDuring those early years, Francis must have thought that consciousness was tractable—if only the right way of thinking was brought to bear on it. Francis's brain was capable of collecting and filing away many disparate data, which he could then combine uniquely and imaginatively, leading to that “dramatic moment of sudden enlightenment that floods the minds when the right idea clicks into place” , p. 141.Over the quarter century he was at the Salk Institute, Francis did propose solutions to some smaller problems in neuroscience  and brouWhen Francis worked on the structure of DNA, he had some simple facts, such as Chargaff's Laws, and means to make point mutations from which it could be determined how function followed structure. But not a single neuroanatomist knew how many neurons actually converged in their input to a particular single cell. No one knew how to eliminate a specific cell type from a circuit— to make a point mutation, so to speak, in the structure of consciousness. His 1979 article in Scientific American, “Thinking about the Brain,” did not have much impact at the time, even when it explicitly described three needed methods: first, a method by which all the connections to a single neuron could be stained; second, a method by which “all neurons of just one type could be inactivated, leaving the others more or less unaltered”; and third, a means to differentially stain each cortical area, “…so that we could see exactly how many there are, how big each one is and exactly how it is connected to other areas.” By the mid-1980s, Francis had realized that these massive holes in our understanding of the most simple brain facts were not being filled. Something needed to be done.Over the twenty years since the RNA Tie Club, molecular biology had matured. Francis actively began encouraging the inclusion of the critical tools of molecular biology in the study of neural circuits and perception; in his thinking, molecular biology was critical to understand how the brain worked because it provided tools. He would encourage junior scientists, postdoctoral trainees, and faculty—all those who had visited him over the years—to think about using these tools. He would give short homilies about the plethora of sub-types of neurons in the retina; would not the cortex be at least as rich in possibilities? Molecular tools could unravel this knot.As we reminisced after Francis's death, we discovered that Francis had spoken with each of us on these molecular methods, across a twenty-year interval. In the mid-1980s, Francis spoke with Ralph, pressing him to consider how he might do highly specific lesions of single neuron types in motion cortex using molecular identifiers. At the time, the only tools imaginable were some sort of killer antibody approach. Twenty years later, Ed recalls Francis continuing to encourage this cross-disciplinary molecular and systems approach. It was absolutely imperative to Francis's vision of the maturation of neuroscience that there would be a conjoining of molecular biology and systems neuroscience. We are sure we were not unique in hearing this call; with how many others had he shared his vision?The science of the mind is a thinker's game. It is chess against the grandest masters, biological evolution and natural selection—and we are just learning to move the pieces. Our viewpoint is often myopic, with our noses pressed against the back row of the chessboard. It is hard to see the pieces, let alone their arrangement or the strategies they are forming. Francis may not have had the overview needed to reveal evolution's gambit, but he knew the moves needed to clear the “tangle of difficulties” , p. 77 tFrancis hoped for simplicity. He wrote, “Curiously enough, in biology it is sometimes those basic problems that look impossibly difficult to solve which yield most easily…. The biological problems that are really difficult to unscramble are those where there is almost infinity of plausible answers and one has to painstakingly attempt to distinguish between them.” . Watson In 1999, Francis felt that gentle and informal direction was not enough. Thus, he convened a meeting of molecular biologists and neuroscientists at the Salk Institute to encourage them to work together. He brought scientists including Tom Albright, Ursula Bellugi, Ed Callaway, Rusty Gage, Steve Heinemann, Terry Sejnowski, Chuck Stevens, and Inder Verma into one room and said it was time to get serious. He reminded them of the advantages of genetic methods for targeting specific cell types within complex neural circuits, and he reiterated the need for methods that could be used to identify, manipulate, and observe neural circuits in action. Not only were methods to be used in transgenic mice, but also methods based on viral vectors were needed to study the visual system of monkeys. From this, a number of initiatives moved forward, with studies ranging from the molecular biology of Williams syndrome to basic molecular tool building .Today the tools are emerging at an ever faster pace, at least in part due to Francis's maneuvers behind the scenes and his encouragement of junior scientists. Time is curing Francis's bout of scientific prematurity . IndividIn 2001, Francis was diagnosed with colon cancer. He realized that the problem of the neural correlates of consciousness might outlast him. Francis was walking with a cane, still not waiting for anyone, nor allowing anyone to wait for him. He continued to find time for new faces in the field and continued to work on consciousness. While he had made many strides forward, he saw the race for him was winding down. He had had his hope for understanding the structure of consciousness. He had laid the groundwork. He decided to encapsulate his ideas in a “Framework” paper with Koch . For manapproach to understanding consciousness; it is both structural and functional, peering forward into the future into what the shape might be. It was clear to his friends and colleagues that Francis was leaving a last testament.Each of the points of the Framework could form a major research initiative. Perhaps they should. But the central point is the As the cancer finally caught up with Francis, he focused on the role of the rarely studied claustrum . He wrotFrancis was doing what he truly loved to his last moments. He needed to be doing science, perhaps more than ever, to take him away from the physical pain that he surely felt. He had built his army. Perhaps none of us even knew we had enlisted, but we had. And he was setting us off on the long march forward into a time that soon would not be for him. Francis died on the cusp of a new age of molecular systems neuroscience. Soon, we will have the tools and the data, but we will not have Francis. Francis had existed between the α of DNA and the Ω of consciousness. And for a man who never believed in the afterlife, he had indeed achieved immortality.


Aging has been shown to slow reflexes and increase reaction time to varied stimuli. However, the effect of Type II diabetes on these same reaction times has not been reported. Diabetes affects peripheral nerves in the somatosensory and auditory system, slows psychomotor responses, and has cognitive effects on those individuals without proper metabolic control, all of which may affect reaction times. The additional slowing of reaction times may affect every-day tasks such as balance, increasing the probability of a slip or fall.2 on a platform upon which a subject stood, were measured in 37 adults over 50 yrs old. Thirteen (mean age = 60.6 ± 6.5 years) had a clinical diagnosis of type II diabetes and 24 (mean age = 59.4 ± 8.0 years) did not. Group averages were compared to averages obtained from nine healthy younger adult group (mean age = 22.7 ± 1.2 years).Reaction times to a plantar touch, a pure tone auditory stimulus, and rightward whole-body lateral movement of 4 mm at 100 mm/sAverage reaction times for plantar touch were significantly longer in diabetic adults than the other two groups, while auditory reaction times were not significantly different among groups. Whole body reaction times were significantly different among all three groups with diabetic adults having the longest reaction times, followed by age-matched adults, and then younger adults.Whole body reaction time has been shown to be a sensitive indicator of differences between young adults, healthy mature adults, and mature diabetic adults. Additionally, the increased reaction time seen in this modality for subjects with diabetes may be one cause of increased slips and falls in this group. Aging slows reflexes and increases the time to react to a number of external stimuli of different modalities -4. What One of the largest implications that an increased reaction time may have is in the area of slips and falls. Falls are incurred by one third of the elderly population and are a common source of morbidity and mortality. EvidencThe aim of this study was to measure and compare reaction times to plantar touch, auditory tone, and whole body lateral movement in subjects over 50 years old with and without diabetes, as well as a group of healthy younger adults under 25 years of age. Subjects with diabetes were expected to have reaction times longer than those of the age-matched controls, while the aged controls were in turn expected to have reaction times greater than those seen in the younger adult group. The implications of the changes in reaction time will be discussed with respect to the central and peripheral nervous system.Subjects included 37 mature adults over 50 yrs old. Thirteen had a clinical diagnosis of type II diabetes made by their primary physician  and 23 did not . The majority of the subjects were recruited from within the Veterans Administration (VA) population at the Overton Brooks VA Medical Center. Reaction times from these groups were compared to a younger adult group  that were recruited through advertising at Louisiana Tech University, and tested at the VA Medical Center. The recruiting, screening, testing and informed consent procedures were reviewed and approved by the local VA Institutional Review Board.Subjects recruited for this study were relatively healthy individuals with no current or past history of severe heart, circulation, or breathing problems; chronic lower back pain or spasms; deformities of the spine, bones or joints (including advanced arthritis); cerebral stroke, spinal cord injuries or other damage to the nervous system; non-healing skin ulcers; advanced diabetes; current drug or alcohol dependence; or repeated falls. Individuals taking any prescription medicine to prevent dizziness were also excluded.Diabetic individuals targeted for this study were those with very early and mild Type II Diabetes. The subject's primary care physician undertook the diagnosis of diabetes. Targeted recruits had all been diagnosed within the last 10 years. All subjects with diabetes were using either diet or oral medication to manage blood sugar levels.Visual, vestibular, muscoskeletal, and cognitive screening was also done to ensure that no undiagnosed problem existed that would prevent subjects from completing the study.Plantar sensory tactile threshold were measured on each sole for all subjects using graded Semmes-Weinstein Monofilaments, which, upon bending, exerted a known force that depends on the filament diameter. Tactile force perception thresholds on the glabrous skin of the feet were determined for the right and left feet using these monofilaments according to standard clinical testing protocol. Stimuli were presented randomly three times at a given location, and, if two of the three presentations were detected, a threshold force was considered determined. Although force is a ratio metric (a measure in which an absolute zero is present and meaningful fractions or ratios can be constructed), the measurement of force by this method is still an ordinal (or rank ordered) type of data. Therefore, non-parametric statistics were used to compare tactile thresholds among test locations on the foot, and between right and left feet, as well as to compare among groups.A certified audiologist at the Overton Brooks VA Medical Center carried out air conduction auditory threshold testing on all mature subjects . Both mature adult groups underwent testing at 1, 2, 4, and 8 kHz in both ears. Average threshold level was recorded in decibels. Using a One Way ANOVA on Ranks, the threshold in each ear was compared to determine any differences in threshold.In addition to this screening, all of the mature subjects underwent clinical surface nerve conduction studies of the lower extremity which were performed at the Neurology Service of the Overton Brooks VA Medical Center by a technician under the supervision of a neurologist. Motor  and sensory nerves  were tested bilaterally. F- and M- latency tests that test the entire lower motor loop (sensory nerve -> vertebrae -> motor nerve) were initially performed to ascertain any problems in the Sherrington's final common pathway . HoweverReaction time was defined as the time between a stimulus onset and a signaled response of the subject. Three different stimuli were presented – touch, tone and platform movement.A manually held, miniature single axis force sensor (Sensotec, Inc) with a 2 mm diameter tip was used by the authors to apply a tactile stimulus to the plantar surface of the big toe of each foot. Since the unloaded sensor force could vary over time or with a change in the position of the sensor, the single axis force sensor was calibrated to a zero state prior to each reaction time test series. A force change of more than 0.01 N was determined to be the trigger for an event. Instructions given to the subjects were to "press the button as soon as you feel the sensory touch the bottom of your foot." Subjects signaled detection of the stimulus via hand held bell button press. The latency between the onset of the rise in applied force measured by the force sensor and the resultant bell-press signal was taken to be the reaction time. Reaction times were measured five times and all trials were averaged. For auditory latencies, a bell tone was presented bilaterally via earphones and a subject signaled by pressing the force sensor with the thumb when he heard the signal. A change of approximately 10 times the sensor's resolution (0.01 N) or greater was determined to be the trigger for the detection of the event. Again, reaction time was averaged over all 5 trials.2 was measured, while a subject stood barefoot on the platform with feet in normal stance. The SLIP-FALLS platform [Finally, a reaction time to a rightward lateral platform movement of 4 mm at 100 mm/splatform  was usedAll data sets analyzed failed a normaility test, prompting the authors to use non-parametric tests. In cases where two groups were compared, a Mann-Whitney Rank Sum Test was used. In cases where three or more groups were compared, a Kurskal-Wallis One Way ANOVA was used. The non-normality of the data also precluded the use of two-way ANOVAs. For all tests, the level of significance used was p < 0.05.Table Because no significant difference p = 0.481) was found between the right and left ears for all groups, the results of the air conduction testing of both ears were pooled and compared between groups. Averages for each group at each frequency (1, 2, 4, and 8 kHz) can be seen in Table  was foun2) is well above the detection threshold of any of the three groups at 4 mm . Hence we have termed this stimulus a Superthreshold stimulus.Determining the minimum acceleration threshold required to detect motion requires special psychophysical test procedures. These procedures, results, and conclusions arising from such testing are complex enough to require treatment in entirely separate papers ,30. In sData was again pooled for both legs because all nerve conduction studies showed no differences between the two legs , yet they do not represent severe slowing. This indicates that those in this study do not have advanced motor deficits, and that the extent of the peripheral neuropathy is significant, yet not severe and debilitating. Also, as expected, sensory nerve conduction velocities were slower than motor nerve conduction velocities, which validate the data.2), plantar touch, and a bell tone were measured in all subjects and can be seen in Table Reaction times to platform movement 4 mm at 100 mm/s00 mm/s2,2, reaction times of all groups are significantly different (p < 0.05) from each other, with reaction times in the adults with diabetes being longest (mean = 777.8 ms), followed by aged matched adults (mean = 623.9 ms). Young adults had the shortest reaction times (mean = 431.0 ms) to movements.For platform movements at 4 mm at 100 mm/sFor the touch modality, reaction times for adults with diabetes are significantly (p < 0.05) longer than both other groups (mean = 353.1 ms). However, reaction times to foot sole touch between young (mean = 216.0 ms) and healthy mature adults (mean = 331.5 ms) were not significantly different.For the tone modality, no significant differences in reaction times were found between groups . For all groups, movement reaction times were significantly longer than the other two modalities (plantar touch and auditory tone), which did not differ significantly.A reaction time measurement includes the latency in the sensory neural code traversing peripheral and central pathways; perceptive, cognitive and volitional processing; a motor signal again traversing both central and peripheral neuronal structures; and finally, the latency in end effector (e.g., muscle) activation. Unless there is a greatly lessened sensitivity or loss of the sensory receptors for a given modality, a stimulus well above perceptual threshold, will, by its very nature of being superthreshold, produce a strong neuronal signal in the peripheral nerve subserving the location stimulated. And, conversely once the volitional decision is made to signal that an event has been detected, the motor output emanating from the spinal cord to the finger that will press the signaling button should likewise be robust.Since reaction time measurements have a central component, any decline, including those seen in normal aging, could indicate the presence of a peripheral and/or central neuropathy. Thus, it becomes difficult to tease out peripheral versus central effects when reaction times are slowed. But if the effect of a peripheral neuropathy is known through knowledge of the change in sensory and motor nerve conduction velocities brought about by the neuropathy, the extent to which the neuropathy will slow the reaction time can be estimated.A lateral platform translational perturbation invokes many senses. These include, but are not limited to pressure and rapidly adapting tactile sensors in the feet and toes, proprioceptive sensors from the ankle and hips , kinesthetic sensors in the muscles , Because of the involvement of all of these "senses," attributing the cause(s) of a slowed platform perturbation reaction time to peripheral and central neuronal changes is difficult. But, if peripheral conduction velocities are known, as are reaction times to more "purer" sensory inputs such as foot sole touch or an auditory tone, then this latter knowledge can be factored into platform reaction time calculations to help tease out peripheral and central effects.At this point, it is instructive to review the results reported in a preceding section of this paper and in Table 1) Subjects with controlled type II diabetes all had mild, but measurable peripheral neuropathies in at least one nerve in the lower limb, while those without diabetes of age >50 had no measurable evidence of neuropathy;2) Subjects with diabetes had increased reaction times to all three test modalities. Touch and Tone reaction times were slightly, but not significantly, higher, while platform reaction time was significantly higher.3) Older adults, whether diabetic or not, had longer reaction times to platform moves and to foot sole touch  than did younger adults, and reaction times to the bell tone did differ between groups, even though those with diabetes had higher auditory air conduction thresholds at every frequency (except 8 kHz) tested than their non-diabetic counterparts.4) Reaction times to platform movement are 200 to 300% longer in all groups when compared to reaction times to touch and tone;Individuals with diabetes often have neurological side effects that affect the peripheral nervous system. However, the increase in whole body movement reaction time seen in adults with diabetes in this study can not solely be related to peripheral nervous system changes due to diabetes. Even when motor nerve conductions slow from 50.0 m/s to 40.0 m/s (as seen in nerve conduction testing here), signal transmission time for a 1 m long nerve increases only 5 ms, which does not account for a 200 ms increase in movement reaction time. An additional slowing has to be occurring in the processing of the signals by the central nervous system.Deficits in the central nervous system (CNS) of those with diabetes may also be seen in cognitive deficits. Dey, et al. found no correlation between the duration of diabetes and cognitive function in those with non-insulin-dependent diabetes less than 18 years old . They hyReaction times to plantar surface touch indicate the extent of peripheral neuropathy in the population of diabetics. The fact that the mature adults with diabetes had increased reaction times to plantar touch is another indication that peripheral neuropathy was present in these subjects. However, we can see that the peripheral neuropathy of these adults with diabetes was not severe through the measurements of the Semmes-Weinstein monofilaments and sensory nerve conduction velocities. This increase may also play a role in the reaction time increase seen in the platform movement. If the subjects were unable to sense the movement for an additional 100 ms, then the 200 ms increase seen in adults with diabetes could be attributed to this sensory reaction time deficit, plus an increase in signal transmission through the nerves of approximately 5 ms, and an unknown cognitive slowing.Auditory reaction times measured here for diabetics and age-matched controls do roughly concur with the one reaction time study that includes diabetics . AlthougFrom this study we can conclude that diabetes does affect reaction times, although the type and severity of the slowing may be related to the difficulty of the task and the prevalence of central and peripheral nerve deficits seen as side effects of diabetes. Auditory reaction times, the simplest of the tasks here with the shortest path between peripheral and central nervous system, did not show any differences in reaction times. When using a test that has a significantly longer path in the peripheral nervous system, such as the reaction time to plantar touch, slightly longer reaction times are seen in the adults with diabetes. When a more complicated task including detecting movement, signal transmission and interpretation, and response was required from the body, as in the platform movement reaction time test, a significant difference in reaction times were seen among all groups. This test takes more fully into account the peripheral nervous system signaling as well as the central nervous system processing and thus is a better overall test to determine deficits in healthy aging and aging individuals with diabetes.We have presented here, in addition to normal auditory and touch reaction times, lateral whole body reaction time, which has been shown to be the most sensitive indicator of differences between healthy young, healthy mature adults, and mature adults with mild diabetes among the modalities tested here. In other studies, we have found that adults with diabetes have substantially higher thresholds than healthy adults to detecting whole body motion . This, iWith the data presented here, it is impossible to determine the relative contribution of peripheral and central neural processes to the slowing seen on the whole body reaction time test. To determine this exact relation, authors are currently working on measures of cognitive processing that may provide more insight.SJR aided in the experimental design, carried out the data acquisition, data analysis and data interpretation, and drafted the manuscript. CJR aided in the experimental design, built the set-up, and aided in the final manuscript. JS completed the statistical data analysis and prepared all the tables and figures.


The key to any protein's function is its structure. Proteins first emerge as a linear strip of amino acids from the cellular protein-manufacturing machinery, and it is this primary sequence that determines a protein's ultimate conformation. Improperly folded proteins—which can gum up cells and, when secreted, tissues—are normally destroyed. But in a wide range of diseases, including prion (from proteinaceous and infectious) diseases and neurodegenerative diseases like Parkinson disease and Alzheimer disease, amyloid fibrils, or plaques—misshapen proteins that aggregate into characteristic rope-like configurations—accumulate in tissue.When amyloid precursors and prions (pronounced PREE-ons) lose their normal conformation, they acquire the ability to infect their neighbors. Like molecular dominoes, the fall of one malformed protein precipitates the downfall of its neighbors, as one protein after another assumes the misshapen form of the first. Any chance of developing methods to contain the expansionist tendencies of these proteins depends on understanding the mechanism of propagation, an area of active research.An abundance of small protein aggregates, called oligomers, is associated with amyloid fiber growth and formation.  Mounting evidence suggests these so-called amyloid intermediates are the “toxic species” underlying amyloid diseases. The steps in amyloid formation, however, are unclear: Must amyloids follow a progression from monomer to oligomer to plaque? That is, are oligomers required for amyloid plaque formation? Using the yeast prion protein Sup35 to study how amyloids form, Jonathan Weissman and colleagues propose a model of amyloid plaque formation and show that it can indeed occur in the absence of the putative toxic oligomers.In yeast, the Sup35 protein forms self-replicating aggregations reminiscent of amyloid formation and prion propagation. Though yeast aren't susceptible to prion diseases, they do assume what scientists call the yeast prion state. Two protein domains called NM together form self-propagating amyloid fibers that give rise to the yeast prion state. Oligomers, which are typically seen when other proteins form amyloids, have also been seen during this process, some of them near NM fiber ends. Weissman's team wanted to know what these oligomers were doing.To investigate the role of oligomers in NM amyloid formation and growth, the researchers explored the relationship between monomer concentration and polymerization progress. Initially, fiber growth rate was tied to the concentration of NM monomers; but as concentrations increased, growth rate was moderated by NM conformational changes caused after binding to the fiber ends. Shaking the samples increased polymerization rate.During polymerization reactions, the authors observed a pronounced pause, followed by an abrupt increase in polymerization rate. Since the length of the pause showed only a weak dependence on the concentration of monomers, Weissman and colleagues explain, this finding could not be explained by a simple model of nucleation polymerization, in which growth occurs monomer by monomer, emerging from a monomer “nucleus.”Instead, Weissman and colleagues' findings support a model in which nucleated monomers initially support fiber growth, fibers undergo fragmentation, and monomers rapidly grow from the broken ends. Weissman and colleagues confirmed that the fibers were growing monomer by monomer by attaching to fragmented fiber ends with fluorescent microscopy, which can detect single molecules.Though the authors do not rule out the possibility that oligomers could attach to the fiber ends as well, their results show that amyloid growth can occur independently of oligomers. Since many of the properties observed in Sup35 polymerization are evident in other amyloid-forming proteins, the model presented here may be shared as well. Future studies will have to explore this question, along with the issues of how oligomers figure into the process and how they cause disease. Weissman and colleagues raise the possibility that creating conditions that favor fiber growth while inhibiting oligomer formation might limit the toxic effects of amyloid plaques. The approaches outlined here should lay the foundation for exploring these questions in higher organisms.


Studying mechanical properties of canine trabecular bone is important for a better understanding of fracture mechanics or bone disorders and is also needed for numerical simulation of canine femora. No detailed data about elastic moduli and degrees of anisotropy of canine femoral trabecular bone has been published so far, hence the purpose of this study was to measure the elastic modulus of trabecular bone in canine femoral heads by ultrasound testing and to assess whether assuming isotropy of the cancellous bone in femoral heads in dogs is a valid simplification.From 8 euthanized dogs, both femora were obtained and cubic specimens were cut from the centre of the femoral head which were oriented along the main pressure and tension trajectories. The specimens were tested using a 100 MHz ultrasound transducer in all three orthogonal directions. The directional elastic moduli of trabecular bone tissue and degrees of anisotropy were calculated.3. The degrees of anisotropy revealed a significant inverse relationship with specimen densities. No significant differences were found between the elastic moduli in x, y and z directions, suggesting an effective isotropy of trabecular bone tissue in canine femoral heads.The elastic modulus along principal bone trajectories was found to be 11.2 GPa ± 0.4, 10.5 ± 2.1 GPa and 10.5 ± 1.8 GPa, respectively. The mean density of the specimens was 1.40 ± 0.09 g/cmThis study presents detailed data about elastic moduli of trabecular bone tissue obtained from canine femoral heads. Limitations of the study are the relatively small number of animals investigated and the measurement of whole specimen densities instead of trabecular bone densities which might lead to an underestimation of Young's moduli. Publications on elastic moduli of trabecular bone tissue present results that are similar to our data.This study provides data about directional elastic moduli and degrees of anisotropy of canine femoral head trabecular bone and might be useful for biomechanical modeling of proximal canine femora. The mechanical properties of canine trabecular bone in the femoral head are important for a better understanding of normal biomechanics of the bone and are needed for assessing changes occurring under pathological conditions like osteoarthritis of the hip, osteonecrosis or Legg-Calvé-Perthes disease of the femoral head. Particularly the elastic moduli are important for finite element modeling of the proximal femur. Several methods have been used in the literature for identifying the elastic modulus of bone, such as mechanical testing -4, combiEight dogs (weight 30–63 kg) were selected that had been euthanized for several medical reasons. From each dog both femora were obtained and were examined by a veterinarian for signs of metastatic malignant disease, Legg-Calvé-Perthes disease, osteoarthritis of the hip or bone necrosis. An x-ray of the whole femur was obtained with the femoral head and the intertrochanteric region placed directly on the film, and the main pressure and tensile trajectories were marked on the image. The bones were kept moist, wrapped in plastic bags and stored at -21°C. Each bone was placed on the x-ray image and the direction of tension and pressure trajectories were marked on the bone according to the x-ray template. An orthogonal coordinate system was defined , and the specimen volumes were calculated. Specimen densities were determined by equation (1):For sonographic testing, a specially designed device with a custom-made ultrasound transducer  was used. An ultrasound frequency of over 2 MHz was chosen for measuring the material properties of trabecular bone tissue. The ultwhere ρ is specimen density, m is specimen mass and V is the specimen volumeThe ultrasound wave runtimes were processed by excluding the minimum and maximum results of the ten subsequent measurements, and the average of the remaining results was calculated. The transmission velocity was calculated by equation (2) and the elastic modulus was determined using equation (3).long is the transmission velocity, s denotes the edge length of the specimen and equals the distance between ultrasound transmitter and receiver which are placed in direct contact with the opposing specimen surfaces, t1 and t0 the time at reception and sending of the ultrasound wave, respectively.where cx,y,z is Young's modulus along x, y and z axes and ρ is the specimen density calculated from equation (1). Clong is the ultrasound velocity calculated from specimen edge length and transmission time (see equation (2))where Ex/Ez; Ex/Ey; Ez/Ey) were calculated and subsequently checked for a significant relationship with specimen density using Pearson correlations.Mean density and mean Young's moduli of the specimens and standard deviations were determined using the statistical software package SPSS 12.0 (SPSS, Chicago, USA). Strong correlations between density and elastic moduli could be expected from equation (3), nonetheless Pearson correlations were calculated for confirmation. The directional elastic moduli were checked for significant differences using one-way ANOVA . The degrees of anisotropy ,21(Ex/Ezx, Ey and Ez) were significant as could be expected from equation (3) (p < 0.005). The degrees of anisotropy ranged from 0.82 to 1.59 and were significantly correlated with specimen density (Table The edge lengths of the bone specimens varied by ± 1 % (± 0.1 mm). Elastic moduli in the bone specimens ranging from 6.3 to 14.3 GPa were found. The elastic moduli in X, Y and Z direction were 11.2 ± 0.4 GPa, 10.5 ± 2.1 GPa and 10.5 ± 1.8 GPa, respectively. Minimum, maximum, mean values and standard deviations of bone sample density and directional Young's moduli are listed in Table This study presents detailed data about mechanical properties of canine femoral trabecular bone tissue and degrees of anisotropy. Despite the strengths of our work, some limitations have to be noted.The correct calculation of the sample volume depends on exactly cubic specimens, but deviations might occur due to errors in the sawing technique. We found a maximum variation of edge length of ± 0.1 mm (1%) in our specimens so this error appears to be negligible.3 for whole bone cylinders after cleaning and 0.65 ± 0.09 g/cm3 for ashed samples which is much lower than our results. The lower densities might be caused by the specimen volume that was used by the authors: they geometrically measured the volume of the cylindrical bone specimens so that a considerable intertrabecular volume is included and bone tissue densities are underestimated according to equation (1). Hence the results of Kang's publication can not be used for comparison or correction of our density data.The apparent densities of the specimens were calculated by weighing whole samples and measuring sample volumes instead of calculating the densities of ashed samples,19 or clIt is noticeable that in our study we found a small relative standard deviation (SD/mean) in apparent densities and Young's modulus in x direction (6.6 % and 3.3 %) although we used femora from a heterogeneous selection of different breeds. However, the relative SD in y and z directions was computed to be 19.5 % and 17.5 %, respectively. It is not clear whether the broader range of elastic moduli in these directions reflects real differences or whether it might be caused by a less accurate positioning of the saw when cutting the specimens from the femoral heads.The significant relationship between the specimen density ρ and directional elastic moduli found in our study was to be expected because Young's modulus was calculated from ρ. This significant relationship was also described in a study using compression testing of bone cubes from human donors  .No significant differences were found between directional elastic moduli (p = 0.34). This result could support the concept of an "effective" isotropic elastic tissue modulus as described by Kabel et al..Several works have been published concerning elastic moduli of canine or human bone. Studies that investigated the apparent elastic modulus of human bone specimens using mechanical testing and finite element models found much lower elastic moduli,21 than Only one study investigating elastic moduli of canine trabecular bone tissue is available to our knowledge: Jorgensen and Kundu  used a 1More studies are available reporting mechanical properties of human trabecular bone. Rho  found anOur study provides detailed data about elastic moduli and degrees of anisotropy of canine femoral bone tissue. No significant differences between directional Young's moduli were found indicating that the concept of an effective isotropy of trabecular bone in canine femoral heads might be a justified simplification. The degrees of anisotropy were highly correlated with specimen densities. The results of elastic moduli are comparable to similar studies of canine and human trabecular bone tissue ,20,26,27TP carried out the statistical analysis of the results, took part in the detailed design of the study and prepared the manuscript. AB designed the study, prepared the bone specimens, conducted the ultrasound measurements and calculated the elastic moduli. UV and AML obtained the canine femora, examined the bone for exclusion criteria and prepared the bone specimens. BAB, IN and HW designed the study concept from a technical and medical perspective, respectively, and corrected the manuscript.


Nonsteroidal anti-inflammatory drugs (NSAID) use may protect against Alzheimer's disease (AD) risk. We sought examine the association between NSAID use and risk of AD, and potential effect modification by APOE-ε4 carrier status and ethnicity.The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD. Subjects comprised 691 AD patients (probands) and 973 family members enrolled at 15 research centers between 1996 and 2002. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. Ethnicity was similarly examined as an effect modifier.NSAID use was less frequent in cases compared to controls in the overall sample . The benefit of NSAID use appeared more pronounced among APOE-ε4 carriers  compared to non-carriers, although this association was not statistically significant. The pattern of association was similar in Caucasian and African Americans.NSAID use is inversely associated with AD and may be modified by APOE genotype. Prospective studies and clinical trials of sufficient power to detect effect modification by APOE-ε4 carrier status are needed. Several cross-sectional ,2, case-The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD sponsored by the National Institute on Aging since 1991. The details of MIRAGE Study data collection procedures, protocols for obtaining family histories, and reports of validity studies of the MIRAGE questionnaires have been published elsewhere. -15 BriefQuestions pertaining to NSAID use were added to the questionnaire in 1996, and the data presented in this report were collected from May, 1996 through May, 2002. Questions about the proband were answered by a surrogate source within the family, typically the spouse or adult offspring. The same information was sought on non-demented first-degree family members of these probands over 50 years of age, usually a sibling or spouse (less commonly parents or children).1020 family members in this analysis claimed to be cognitively normal, or were reported by family informants to be dementia-free. Of these, 982 were evaluated using the modified Telephone Interview of Cognitive Status (mTICS) ,18, and nonsteroidal anti-inflammatory medication (e.g. Advil, Motrin, etc.) on a daily basis for more than 6 months?" No distinction was made between aspirin and other classes of NSAIDs. For proxy reporting about a relative with AD, the question substituted "your relative" for "you". For any affirmative answer, a follow-up question asked for the dates at which the medications were first used and the names of all NSAIDs that had been used.To elicit information on prior NSAID use, the following question was asked: "Have you ever taken a A discrete "index date" was established within each family corresponding to the earliest date that the family or medical records reported AD symptoms to have begun in the proband. Subjects from each family (whether AD cases or non-demented family members) were considered to have been exposed to NSAIDs only if the starting date for NSAID use preceded this index date by at least one year. Age represented the age of cases and of non-demented relatives at the index date, and was treated as a continuous variable.As shown in Figure Analyses were performed using SAS version 8.2. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. Crude odds ratios were computed in the first instance, followed by adjusted estimates using generalized estimating equation (GEE) models  to accouWe stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. In addition, we formally evaluated these associations by adding an interaction term (ε4 * NSAID use) to the GEE model. Ethnicity was similarly examined as an effect modifier.Characteristics of the 1664 subjects are listed in Table Sixty-six out of 973 non-demented relatives (6.8%) and 24 of 691 cases (3.5%) reported previous NSAID use . After adjustment for age, sex, educational level, and ethnicity, the odds ratio (OR) of NSAID use among AD cases compared to non-users was 0.57 (95% CI = 0.35–0.93); it was 0.64 (95% CI = 0.38–1.05) when APOE carrier status was added to the GEE model  than non-carriers . However, formal evaluation of the interaction between NSAID use and APOE-ε4 carrier status did not reveal a significant difference (p = 0.40). The association between NSAID use and AD risk was similar among Caucasian and African Americans (data not shown).This study supports the findings of previous reports -6 suggesThe MIRAGE Study includes the largest number of well-characterized AD cases and family controls to date, and this large sample size permits adjustment for important potential confounders, as well as the power to examine effect modification by APOE genotype and ethnicity. The subjects without dementia were first-degree family members of AD cases, providing some degree of informal matching on age, socioeconomic status, and health-seeking behavior.However, these results must be interpreted in light of some methodological limitations. Data on NSAID use was collected with a single retrospective question that did not distinguish between aspirin use and non-aspirin NSAID use. Moreover, while non-demented participants reported on themselves, a proxy historian reported on most of the demented individuals.Differential reporting is a potential source of bias in a study that uses self-report on most of the non-demented subjects, yet relies on surrogate respondents for all of the subjects with AD. Asymmetric data collection is difficult to avoid when cases are cognitively impaired, but may be more accurate than expected in AD patients where the surrogate historian has a long association with the subject. We addressed this potential bias by performing an independent validation study to determine the accuracy of surrogate information on a number of questions, including the same questions used in this report about NSAID use.  This stuThis result is consistent with those of prior studies which found a similar association despite differences in study design . Differential recall could also give rise to this observation. However, these explanations are unlikely because subjects were not selected on the basis of APOE genotype. It is also possible that APOE-ε4 carrier status is a proxy for differentially distributed unmeasured confounders related to NSAID use such as inflammatory disease processes.Alternatively, our results imply that NSAID use affects AD risk differently between APOE-ε4 carriers and non-carriers. For example, because ε4 carriers are inherently more vulnerable to AD, there is a greater opportunity for attributable risk reduction. This explanation does not imply biological interaction between NSAIDs and ε4. On the other hand, the ε4 isoform may have greater pro-inflammatory properties  and ε4 iExamination of this finding in prospective studies and clinical trials of sufficient power  declare that they have no competing interests.Study concept and design (LAF, RCG, LAC); acquisition of data (LAF, RCG, MIRAGE investigators); analysis and interpretation of data (AGY, MH); drafting of the manuscript (AGY, RCG, LAF); critical revision of the manuscript for important intellectual content (AGY, LAF, RCG, LAC); statistical expertise (LAC); obtained funding (LAF, RCG, LAC, MIRAGE investigators). All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor α (RXRα), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRα are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRα was a common mechanism underlying the negative hepatic APR.Nuclear RXRα protein levels were significantly reduced (~50%) within 1–2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRα was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRα partner, the retinoic acid receptor (RARα), was unaffected by LPS. A potential cell-signaling modulator of RXRα activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1–2 hours, coincident with maximal levels of cytoplasmic RXRα. RNA levels of RXRα were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRα were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRα-containing heterodimer pairs.The subcellular localization of native RXRα rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRα-regulated genes in inflammation. Ntcp/Slc10a1) and organic anion transporting proteins (Oatp1/Slc21a1), as well as the canalicular multispecific organic anion exporter (Mrp2/Abcc2) and the bile salt export protein (Bsep/Abcb11). Transcriptional down-regulation of the principal hepatic bile acid importer, Ntcp contributes to the reduction in bile acid uptake by hepatocytes in inflammation, whereas reduced Mrp2 expression leads to impaired excretion of conjugated bilirubin, glutathione and other organic anions into bile [LPS, a major constituent of the outer membrane of gram-negative bacteria, potently stimulates host innate immune response . LPS-indnto bile ,6.et al. showed that LPS-mediated suppression of Ntcp RNA was almost completely blocked by pre-treatment with anti-IL-1β specific antibodies, indicating that the cholestatic effects of LPS on the expression of this gene may be primarily mediated by the cell signaling pathways initiated by this one cytokine [Ntcp transactivator, RXRα:RARα, with consequent down-regulation of Ntcp promoter activity [Recent reports have provided insights into the link between inflammation-mediated cell signaling and regulation of bile acid homeostasis in the liver. Geier cytokine . We haveactivity . Finallyactivity -11. How activity .et al [Phosphorylation of nuclear receptors (NRs) is a rapid and potentially powerful means of regulating NR activity, that, depending upon the NR, can affect transcriptional activity, protein stability, sub-cellular localization, protein-protein interactions or DNA binding activity ,13. Phoset al  and the et al . In contet al ,17. CleaNR ligands and extracellular signal-mediated pathways can alter subcellular NR localization, some of which involves phosphorylation-dependent mechanisms ,13,18-20RXRα and six of its heterodimeric partners highly expressed in liver were analyzed after LPS treatment: RXRα, RARα, FXR (farnesoid X receptor) and PPARα (peroxisome proliferator-activated receptor) RNA levels were stable, whereas CAR (constitutive androstane receptor) and PXR (pregnane X receptor) RNA levels were markedly suppressed and LXR (liver X receptor) RNA was elevated. Hepatic RNA levels of multiple RXRα target genes whose expression depends upon adequate nuclear levels of RXRα were significantly reduced by LPS. This is likely to be a consequence of reduced nuclear binding activity of RXRα heterodimer pairs. Notably, the reduction in RXRα nuclear protein levels (~50%) quantitatively correlated with the reduction in RNA levels of RXRα target genes. Taken together, these studies indicate that post-translational modification and cellular re-distribution of RXRα coinciding with induction of cell signaling is a novel, broad-ranging, and rapid mechanism contributing to the negative hepatic APR phenotype in the inflamed liver.In these studies, we sought to determine whether alterations in RXRα-dependent hepatic gene expression seen in inflammation may be related to nucleo-cytoplasmic re-distribution of RXRα. LPS treatment resulted in the activation of hepatic JNK coinciding with marked reduction in nuclear RXRα levels, and the rapid appearance of RXRα in the cytosol. RNA levels of In order to determine the in vivo role for LPS-induced activation of hepatic JNK, we first established a time course for JNK activation, by measuring phospho-JNK and phospho-c-Jun levels. Liver whole cell extracts were prepared at various time points from 1–16 hours after injection with either LPS (2 μg/g bw) or vehicle  Fig. . PhosphoTreatment of HepG2 cells or primary rat hepatocytes with IL-1β leads to JNK-dependent repression of RXRα :RARα nuclear binding activity, with the consequent down-regulation of target gene expression . HoweverTo confirm the intracellular localization of RXRα in liver, immunofluorescence staining was carried out in formalin-fixed liver tissues prepared from mice 1 h after saline or LPS injection Fig. . In LPS-RXRα RNA expression, as seen in response to higher doses of LPS [RXRα, six of its heterodimeric partners and SHP–all known to be involved in hepatic gene expression [RXRα, RAR, FXR and PPARα were unchanged, whereas PXR and CAR RNA levels were reduced and LXRα RNA levels were increased , Bsep (FXR), Mrp2 (CAR, FXR, PXR), Cyp3A11 (CAR, PXR), Abcg5 (LXRα) and Lfabp (PPARα)  Fig. . RNA leviewed in ).The orphan nuclear receptor SHP  is known to repress the activities of RXRα and other NRs ,29. One In order to determine if reduced nuclear RXRα protein levels leads to impaired DNA binding activity of RXRα and its partners, electrophoretic mobility shift analyses were performed. Nuclear extracts were prepared from livers of saline or LPS-treated mice and incubated with oligonucleotides containing canonical DNA elements scanning Type II NR binding sites–direct repeats of the hexad AGGTCA, separated by 1 to 5 nucleotides (DR1-5), or an inverted repeat separated by 1 nucleotide (IR1) ,31. As TThe negative hepatic APR is characterized by suppression of hepatic gene expression in response to inflammation and is well-modeled by LPS administration ,33. We hNtcp gene expression, thus implicating JNK to be a central player in inflammation-induced cholestasis. Most evident in the 1 hour sample are high molecular weight forms of RXRα, consistent with covalent post-translational modification . In transfected cells, nerve growth factor (NGF)-induced phosphorylation of the orphan nuclear receptor NGFI-B (Nur77) resulted in the translocation of RXR-NGFI-B complex out of the nucleus, indicating that distribution of RXR in these cells was regulated by NGFI-B [The interrelationship and roles played by JNK and phospho-RXRα are neither readily nor definitively explored in an in vivo model, especially using such a broadly-acting inflammatory agent like LPS. Hepatocytes and liver-derived HepG2 cells in culture respond to LPS-induced cytokines like TNFα and IL-1β by suppressing the expression of negative hepatic APR genes ,24,26,36y NGFI-B . The daty NGFI-B . FinallyOverall, we conclude that RXRα is rapidly exported out of the nucleus in response to LPS. RXRα, as an obligate heterodimer with other class II NRs, regulates the expression of a broad array of genes involved in critical metabolic pathways in the liver, many of which are impaired during the negative hepatic APR. This helps explain how inflammation-induced signaling can lead to rapid, diverse and multiple alterations in hepatic gene expression, which has implications for future therapeutic targets of both acute and chronic liver diseases.Salmonella typhimurium) was purchased from Sigma Chemical Co. (St. Louis, MO) and freshly diluted to the desired concentration in pyrogen-free 0.9% saline before injection. Anti-JNK (#9252), phospho-JNK (#9251) and phospho-cJUN antibodies (Ser 63) (#9261) ; anti-RXRα (D-20) (#sc-553) and anti-RARα antibodies (#sc-551) (Santa Cruz Biotechnology, Santa Cruz, CA) were used according to manufacturer's instructions. [γ-32P]ATP was obtained from PerkinElmer Life Sciences (Boston, MA). Oligonucleotides were obtained from Sigma Genosys and Synthegen, Houston, TX. All reagents for real-time PCR were purchased from Applied Biosystems (Foster City, CA).LPS  in saline or saline alone. LPS in this dose range has been shown previously to induce cholestasis, maximally inhibit bile acid uptake, and significantly reduce Ntcp mRNA from 12 to 16 hours after injection, while not inducing hepatic damage [Adult male (8–10 weeks) C57BL/6 mice (20–25 g) were purchased from Charles River Laboratories, (Wilmington, MA). The animals were maintained in a temperature- and humidity-controlled environment and were provided with water and rodent chow ad lib. Mice were given intraperitoneal injection with 2 μg/g body wt LPS  antibody and fluorescein isothiocyanate (FITC)-labeled secondary antibody and nuclei was stained with 4'-6-diamidino-2-phenylindole (DAPI). Visualization was performed with a Deltavision Spectris Deconvolution Microscope System (Applied Precision, Inc.).et al.[32P end-labeled oligonucleotide as described previously [Nuclear extracts were prepared according to Timchenko et al. with somet al.. 10 μg oeviously . The oli® Universal PCR master mix. PCR thermocycling parameters were 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 s, and 60°C for 1 min. Quantitative expression values were extrapolated from standard curves and were normalized to GAPDH. The sequences of the primers and probes were obtained from the literature [Total RNA was isolated from mouse liver tissues using the RNaesy kit from Qiagen. cDNA was synthesized from 7.5 μg of total RNA using the ProSTAR™ First-Strand RT-PCR Kit (Stratagene, La Jolla, CA). Real time quantitative PCR (RTQ-PCR) was performed using an ABI PRISM 7700 Sequence Detection System instrument and software (Applied Biosystems, Inc., Foster City, CA). Briefly, each amplification reaction (50 μl) contained 40–200 ng of cDNA, 300 nM of forward primer, 300 nM of reverse primer, 200 nM of fluorogenic probe and 25 μl of TaqManterature  or purchThe abbreviations used are: RXR, retinoid X receptor; RAR, retinoic acid receptor; FXR, farnesoid X receptor; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; CAR, constitutive androstane receptor; LXR, liver X receptor; SHP, small heterodimer partner; NR, nuclear receptor; GR, glucocorticoid receptor; PR, progesterone receptor; VDR, vitamin D receptor; JNK, c-Jun N-terminal kinase; AP-1, activator protein-1; Ntcp, sodium/taurocholate cotransporting polypeptide; Bsep, Bile salt export pump; Mrp2, multidrug resistance associated protein 2; Lfabp, liver fatty acid binding protein; Cyp3A11, cytochrome P450 3A11; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APR, acute phase response; DR, Direct Repeat; IR, Inverted Repeat; FITC, fluorescein isothiocyanate; DAPI, 4'-6-diamidino-2-phenylindole.


Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs).The genome of gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit.On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of  Parachlamydia acanthamoebae is an obligate intracellular bacterium naturally infecting free-living amoebae [Acanthamoeba spp. recovered from the nasal mucosa of healthy volunteers [Parachlamydiaceae have been found within about 5% of Acanthamoeba spp. and once within Hartmanella vermiformis [Parachlamydiaceae are about 14% different from those of both genera Chlamydophila and Chlamydia [Chlamydophila sp. and Chlamydia sp. is 6% only, it clearly appears that the speciation between the two latter occurred after the divergence between Parachlamydiaceae and Chlamydiaceae.First described in 1997,  amoebae . It was miformis ,3. The 1hlamydia ,3. SinceChlamydiales, Parachlamydiaceae can present two developmental stages: the reticulate body, a metabolically active dividing form, and the elementary body, an infective stage; the crescent body is another infective form, not observed in Chlamydiaceae [Like other ydiaceae . Differeydiaceae . Dependiydiaceae .Parachlamydiaceae [Parachlamydia serologies were shown to be associated with a febrile epidemic [Parachlamydia-related bacteria as agents of inhalation pneumonia is further suggested by the temperature-dependent release of the bacteria from their amoebal reservoir [Parachlamydia [Chlamydia-like organisms within human macrophages [There is a growing evidence of the human pathogenicity of ydiaceae . For insepidemic , communiepidemic , and inhepidemic . The roleservoir . PCR amphlamydia -12. The rophages  is an adet al. [Parachlamydia-related UWE25 contributed much to the understanding of the evolution of chlamydiae. Indeed, they demonstrated that major virulence mechanisms of Chlamydiaceae such as the Type Three Secretion System (TTSS) and the Chlamydial Protease-like Activity Factor (CPAF) are also encoded by the chromosome of the evolutionary early-branching Parachlamydiaceae UWE25. Genome analysis of the parachlamydial endosymbiont also identified Open Reading Frames (ORFs) homologous to Type Four Secretion Systems (TFSS) and characterized by a high G+C content, suggesting that they result from an horizontal transfer. Based on their annotation revealing the apparent absence of genes necessary for DNA transfer, Horn et al. [Horn et al. , by sequn et al.  proposedTo date, numerous genomic islands (GIs) were already identified along whole chromosomal sequences of various bacterial species. For instance, 140 GIs are described in the Islander database, including GIs of proteobacteria, firmicutes, actinobacteria and cyanobacteria . Thus, wloci are generally used as insertion sites by GIs for their chromosomal integration [GIs are genetic elements which length vary from 10 to 200 kb and are inserted in a chromosome after a lateral transfer occurring, in some instances, between phylogenetically-distant microorganisms. Their heterologous origins are generally evidenced by a G+C content different from that of the remaining bacterial chromosome and by the presence of various mobility genes (i.e. involved in transposition, transduction or conjugative transfer), that are occasionally source of GI instability ,17. Theyegration -18. SincParachlamydia-related UWE25 chromosome, we observed a G+C-rich region  according to the nomenclature used in the Islander database [Using standard G+C content analyses of n Figure  and 1B, e et al. . Using te et al. , we weret Figure , Table 1database . Interestingly, this GI can be divided into clearly distinct regions, according to their G+C content Figure , Table 2gly-tRNAs in tandem. Composed of 28 ORFs, this unit exhibits a G+C content (36.4%) similar to that of the remaining of the genome of UWE25 (1931 ORFs, 36.1%). Sixteen homologs to these 28 genes (57%) were found in databases, 12 of them (75%) exhibiting a best score in BLAST analyses with a Chlamydiaceae ORF , four of them being identified as mobility genes (three bacteriophage-related genes and one putative transposase encoding gene).Characterized by a low G+C content (34.1%), the 2rd module (19 kb), exhibiting the second highest G+C content of the UWE25 genome (40.9%), comprises 21 ORFs. Some of these genes were identified as tra genes by Horn et al. [tra operons encoding the TFSS previously described in proteobacterial genomes  is composed of 13 ORFs. All these ORFs were previously annotated by Horn et al. [Presenting a low G+C content (33.4%), the 4n et al.  as encodh module 0 kb is cth module (6 kb). Its G+C content is one of the highest of the UWE25 chromosome (41.8%). By BLAST analysis, this protein exhibits the strongest similarity with the human Nod3 protein.A single large protein is encoded along the 5th module (12 kb) is characterized by a low G+C content (33.3%). This unit is composed of 13 ORFs, the first ORF encoding a product similar to the Death on cure (Doc) protein of P1 bacteriophage. Two ORFs code for proteins involved in DNA metabolism and an additional ORF encodes a putative transposase.The 6th module is short (2 kb) and present a G+C-rich unit (38.7%). Five of the six ORFs of this unit encode a probable resolvase, three putative transposases and a phage-related Doc protein. The final direct repeat is located at the end of this module. With the only exception of the phage-related protein, all other ORFs of the 7th module appear to be similar to gamma-proteobacterial proteins, possibly explaining the observed different signal in the G+C content analysis.The 7tra genes showed a genetic unit unable to transfer DNA [tra genes, and compared the general organization of this tra unit with other genetic elements encoding TFSS genes [tra unit displays a striking colinearity with tra operons encoding F-like conjugative DNA transfer system, especially to those of the F and pNL1 plasmids of Escherichia coli and Novosphingobium aromaticivorans, respectively  and involved in sex pilus retraction and mating pair stabilization [tra unit are devoted to DNA transfer. With the only exception of traG, these genes are not present on P-like and I-like plasmids, reinforcing the close relationship prevailing between the UWE25 tra unit and their F-like plasmids counterparts.The functions of genes encoded by GIs may be related, among others, to pathogenicity such as the ability to exploit the host intracellular environment. Since no genetic system has been described for any obligate intracellular chlamydiae, we investigated the putative functions of this GI by bioinformatics. We focused our analyses on the TFSS, for which a previous annotation of the sfer DNA . Using dSS genes . The UWEy Figure . All homlization  are prestra unit clusters within F-like TFSSs, confirming that it may function as a F-like conjugative system. Drawn as an UPGMA tree . An omit test performed on this tree confirms that the results are robust: with one exception (involving the deep branching of one cluster on one tree), all 11 trees were congruent in all their nodes. Figure p-distances (the proportion p of nucleotide sites at which two sequences are different, taking into account the proportion of transversion- and transition-substitution rates) of nucleotide sequences of the concatenated traA, traK, traB, traV, and traC genes. A similar topology is observed with (i) neighbor-joining- and minimum evolution trees inferred using the Kimura-corrected p-distances and (ii) UPGMA, neighbor-joining- and minimum evolution trees performed on p-distance of the whole coding sequences of the concatenated tra genes . Moreover, the BLAST analyses of the 21 ORFs of the third module, encoding the tra genes, show that most ORFs of this unit (15/21) are of proteobacterial origin. However, since six of them present the highest similarity to alpha-proteobacterial genes and six others to gamma-proteobacterial genes, a more precise origin of the parachlamydial tra unit could not be precisely defined by this first approach.Our BLAST analyses  reveal tgly-tRNA at the proximal end of the GI of UWE25 is consistent with a close relatedness between this GI and proteobacteria: out of 14 GIs described in the Islander database of Mantri et al. [gly-tRNA (14/140), 12 of them (86%) were sequenced in a proteobacterial genome. No GI of Gram-positives described in the Islander database are inserted in a chromosome within a gly-tRNA gene. Again, a precise proteobacterial origin could not be proposed, because the distribution of gly-tRNA genes in alpha- (4/22) and gamma-proteobacterial (8/72) GIs is not significantly different: by including only the non-redundant GIs, the distribution of gly-tRNA genes in alpha and gamma-proteobacterial GIs is 2/20 and 7/71, respectively.The presence of i et al. ,22 insertra units also failed in assigning a precise origin to the UWE25 tra unit since it branched near the alpha- and gamma-proteobacterial tra operons  on similarity analyses demonstrating the presence of all genes encoding proteins used during a DNA transfer, (ii) on phylogenetic analyses of tra unit genes and, (iii) on comparison of gene order. These analyses clearly demonstrate that the UWE25 tra unit is strongly more related to F-like conjugative system than to P-like and I-like secretion systems. The significant bootstraps of all trees obtained by standard gene phylogeny and their congruent topologies with others obtained by the gene order breakpoint analysis not biased by codon usage homing, strongly support the validity of these analyses confirming the F-like conjugative nature of the parachlamydial tra unit. Thus, our model significantly differs from the other proposed by Horn et al. [traA, traL, traK, traV, and concluded that the UWE25 tra unit is involved in protein export, and not in DNA transfer.The 3n et al. , who didth module presents a nucleotide composition similar to the tra unit and is composed of a single high G+C 6-kb gene, whose product is similar to the human Nod3 protein. The Nod (Nucleotide-binding oligomerization domain) proteins are members of a family that also includes the apoptosis regulator Apaf1 (Apoptotic protease activating factor 1) and plant disease-resistance gene products [The 5products . The funnd, 4th, and 6th modules are similar, explaining the observed similar negative slope of the residual G+C curves. Moreover, these three modules encode phage-related proteins and proteins involved in DNA metabolism. These modules probably involved in mobility might have a common origin, the ancestral single phage module being currently separated in three pieces by the presence of the tra unit and of the Nod3-like protein encoding gene.The nucleotide G+C composition of the 2th module echoes those of the tra unit (3rd module) and of the Nod3-like protein (5th module). The 7th module encodes a transposition resolvase and three transposases similar to gamma-proteobacterial homologs. With the only exception of the phage-related Doc protein, that has an homolog at the beginning of the sixth module, and that might be located there after transposition, the 7th module appears thus to have a different origin than the 2nd, 4th and 6th modules, though also encoding mobility genes.The positive slope in the G+C analysis of the 7tra unit along the sequences of UWE25 is the first evidence of a putative conjugative system in chlamydiae. If conjugation occurs, it most probably takes place within free-living amoebae, that may contain several hundreds of Parachlamydia bacteria tightly packed in their vacuoles [The presence of a F-like vacuoles . Such a tra units/operons in the parachlamydial UWE25 and in proteobacteria could be explained by an emergence of this unit in a common ancestor of both clades, and by its subsequent loss in Chlamydiaceae. Another evolutionary scenario is that the tra unit was acquired from a proteobacteria by a Parachlamydiaceae in a common amoebal vacuole. Since the tra unit is absent from all sequenced Chlamydiaceae genomes, this transfer would have occurred after the divergence of Parachlamydiaceae and Chlamydiaceae, at a time when Parachlamydia was already an intracellular bacteria. An intra-amoebal transfer of this GI is supported by the permissivity of free-living amoebae to proteobacteria [tra, further analyses have to confirm whether this GI module was acquired from an alpha-, beta-, or gamma-proteobacteria unit. We hypothesize that the F-like parachlamydial tra unit has been brought by a lateral transfer from a proteobacterial genome. This hypothesis is strongly supported by the cumulative GC skew analysis [Chlamydiales. Sequencing additional genomes of environmental chlamydiae, that present a large biodiversity [The presence of bacteria , and by analysis -30 produanalysis . The genParachlamydiaceae bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired by a Parachlamydiaceae from phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia [Chlamydiales.We showed that a GI present on the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of neumonia , the PamParachlamydia-related endosymbiont UWE25, and thus not to the Acanthamoeba sp. strain UWE25 from which the parachlamydial endosymbiont UWE25 was recovered [et al. recently proposed UWE25 as the type strain of a new bacterial species: Protochlamydia amoebophila [The genome sequence of UWE25   = knn is the position of the center of the nth window. The residual cumulative G+C content curve GC' can then be drawn as a function of the position of each window center:where GC'n = GCn - knZhang and Zhang  recentlytra genes previously identified by Horn et al. [The perfect tandem repeats identification was first performed using the EQUICKTANDEM software (Richard Durbin, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK)  on a 200n et al. . The dupn et al. , with thet al. [traA, traL, traK, traV, re-annotation of the UWE25 tra unit was necessary for phylogenetic analyses. We used i) the genes of F-like plasmids encoding the following tra genes, i.e. traA, traK, traB, traV, traC, and ii) the corresponding ORFs of P- and I-like plasmids [trbC/VirB2, trbG/VirB9, trbI/VirB10, trbH/VirB7, trbE/VirB4 of P-plasmids and traX, traN, traO, traI, traU of I-plasmids, respectively. The genes were concatenated to obtain a single nucleotide sequence and aligned with CLUSTALW ([p-distances (the proportion p of nucleotide sites at which two sequences compared are different) and Kimura corrected p-distance (correction for the rates of transition and transversion) with Unweighted Pair Group Method with Arithmetic Mean (UPGMA), neighbor-joining, and minimum evolution methods. To prevent alignment biases, trees were drawn using the complete deletion option implemented on MEGA 2.1.Since Horn et al.  did not plasmids , i.e. trLUSTALW  by Blanchette et al. [tra unit of UWE25 and the tra operons reviewed by Lawley et al. [Oi and Oj containing homologous genes proposed by Blanchette et al. [tra operons: instead of counting the number of minimal breakpoints existing between two tra operons, a distance was estimated by measuring the proportion of conserved gene pairs between both genomic entities. Next, a comparison matrix is established by calculating the distance for each pairwise comparison. Finally, a dissimilarity matrix is obtained by subtracting each distance from 1.To quantify the inversion and transposition events leading to the current organization of e et al.  was usedy et al. . The dise et al.  was sligOi encodes sequentially four genes (a, b, c, and d) and the operon Oj, six genes , the gene order breakpoint analysis reveals that two gene pairs are conserved: ab and cd. The dissimilarity distances existing between the operons i) Oi and Oj, and ii) Oj and Oi would be: 1-(2/3) = 1/3 and 1-(2/5) = 3/5, respectively.For instance, if the operon omit test [From the square dissimilarity matrix, phylogenetic trees were drawn. Three different distance-matrix analyses were used: the UPGMA, the Fitch-Margoliash- and the minimum evolution methods. To assess the robustness of the tree, an mit test  was perftra unit and performed all BLAST analyses. FC and LG delimited the GI by direct repeat analyses. GG drew the phylogenetic trees of the concatenated tra genes. After developing the residual cumulative G+C content analysis used for a software development, LG performed the G+C content analyses and the gene order comparison of tra units by the gene order breakpoint analysis. CAR established the correlation existing between the tRNA genes and the GIs according to taxonomy and coordinated the team work. GG wrote the first draft of the paper. All authors improved the manuscript and approved its final version.GG and CAR initiated the project. GG and FC reannotated the parachlamydial Chlamydiaceae are presented. Direct repeats (DR), gly-tRNA genes and limits of each modules of the GI are highlighted.Supplementary table. Results of BLAST ,36 analyClick here for filetra unit is phylogenetically closely related to F-like DNA conjugative tra operons: (A) UPGMA-, (B) Neighbor-joining-, and (C) minimum evolution-trees comparing p-distances and Kimura corrected p-distances of nucleotide sequences of the concatenated traA, traK, traB, traV, and traC genes (the UPGMA tree comparing the Kimura corrected p-distances of tra genes, shown in Figure p-distances, the tra unit of UWE25 is clustered with tra operons of gamma-proteobacterial F-like plasmids: bootstrap values of 96% and 92%, respectively, support the node separating the concatenated tra genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmids, from their closest relative R27 plasmid. Similarly, in neighbor-joining and minimum evolution analyses of the Kimura corrected p-distances, bootstrap values of 94% and 91%, respectively, support the node separating the concatenated tra genes of the chromosomal UWE25 and the R27 plasmid, another gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids.Supplementary figure. Phylogenetic analyses suggest that the UWE25 Click here for file