Curculio sikkimensis (Curculionidae), a generalist seed predator of Fagaceae plants living in both deciduous oak and evergreen forests of Japan. Its genetic structure was compared to that of another host-specific seed predator, C. hilgendorfi, inhabiting only evergreen forests.Climatic changes during glacial periods have had a major influence on the recent evolutionary history of living organisms, even in temperate forests on islands, where the land was not covered with ice sheets. We investigated the phylogeographical patterns of the weevil C. sikkimensis from 11 plant species of three genera, Quercus, Lithocarpus, and Castanopsis. An analysis of molecular variance revealed that a large proportion  of the total genetic variance could be explained by differences between two geographical regions, the southwestern and northeastern parts of the main islands of Japan. In contrast, no significant genetic differentiation of the weevil was observed among vegetation types of their utilized host plant species. The phylogeographical patterns of the generalist and the host-specific seed predator exhibited a congruent genetic boundary in the Chugoku-Shikoku region.We examined 921 bp of mitochondrial DNA for 115 individuals collected from 33 populations of C. sikkimensis. The geographical patterns of genetic differentiation in the Chugoku-Shikoku region observed in the two types of Fagaceae-associated Curculio in this study have also been observed in several plant species growing in warm and cool temperate zones of Japan. The occurrence of this common pattern suggests that deciduous oak and evergreen forests of Japan survived together, or adjacent to each other, in small refugia during glacial ages, in the southwestern and northeastern parts of the main islands, although these two types of forests are presently distributed in cool and warm temperate zones of Japan, respectively.Our results suggest that geology and historical environment have contributed to shaping the present genetic structure of  The current distribution of biological diversity cannot be understood without information about how organisms responded to historical geological and climatic conditions. In particular, the glacial-interglacial cycles of the Quaternary period may have played an important role in shaping the distribution of biodiversity among current populations, even in warm temperate zones, where the land was not covered with ice sheets ,2. The gPhylogeographical patterns estimated from the extant genetic variation of organisms have proved highly informative in recovering the postglacial demographic histories of species ,4. AnothPalynological evidence indicates that broadleaved deciduous and evergreen forests in Japan were subjected to cold periods at least four times during the Quaternary ,2. DurinCurculio oviposit in seeds that are still on the tree. Females use the rostrum to excavate a hole in the seeds , and C. hilgendorfi is a host-specific seed predator of Castanopsis. The other species are associated with several species of deciduous Quercus. Curculio sikkimensis and C. hilgendorfi are sometimes sympatrically distributed in evergreen forests.Five species of Curculio species, C. sikkimensis and C. hilgendorfi, validated each clade with high bootstrap support  Do the mtDNA haplotypes of C. sikkimensis form host races? If C. sikkimensis exhibits some host specialization, the genetic differentiation of mtDNA in C. sikkimensis should be observable among their host Fagaceae species, among host plant genera, and between deciduous and evergreen host plants . 2] Do the phylogeographical patterns of Curculio species enable us to reconstruct the glacial and postglacial history of their associated temperate forests in Japan? Given that host plant species inhabit both broadleaved deciduous oak and evergreen forests, an investigation of the generalist predator C. sikkimensis provides an understanding of the biogeographical history of both deciduous oak and evergreen forests in Japan. However, the phylogeographical information of the specialist C. hilgendorfi reflects the history of evergreen forests. We compared the phylogeographical results of the generalist C. sikkimensis and the specialist C. hilgendorfi as well as those of the plant species growing to examine whether the evolutionary histories of their associated deciduous oak and evergreen forests are similar.The purpose of this study was to assess the phylogeography of the generalist acorn weevil Quercus, Lithocarpus, and Castanopsis infested with C. sikkimensis from 33 localities in Japan  were amplified using the following PCR primers: COI-1729F, 5'-GGATCACCTGATATAGCATTCCC-3' and COI-2764R, 5'-CCTAAAAAATGTTGTGGGAAAAAGG-3' modified from  were aligned using Sequence Navigator software (Applied Biosystems).We analyzed sequence data for 115 ied from . Amplifih), nucleotide diversity (π), and Tajima's D . How. HowC. hC. sikkimensis and C. hilgendorfi. Castanopsis sieboldii var.sieboldii, which is the major host plant species of C. hilgendorfi, is rarely distributed around the Seto Inland Sea. In the case of the host-specific seed predator C. hilgendorfi, the lack of distribution of the main host plant species around the Seto Inland Sea may have prompted genetic differentiation between C. hilgendorfi on the Pacific Coast and those on the coast of the Sea of Japan. In contrast, C. sikkimensis is a generalist seed predator of Fagaceae plants and could migrate across the Seto Inland Sea using various Fagaceae plant species in the Chugoku and Shikoku regions. This polyphagous habit allowed C. sikkimensis to cross the Seto Inland Sea and also may have generated the intermediate CS clades. A more detailed history of the area around the Seto Inland Sea can be estimated by comparing the phylogeographical patterns of various organisms with diverse geographic distributions and life histories.Two differences in phylogeographical patterns were observed between Castanopsis and Lithocarpus, as well as the possible introgression among Quercus species [Quercus; the geographic distribution of cpDNA haplotypes in deciduous Quercus indicated that one of the haplotypes is restricted to eastern Japan [Little phylogeographical data are available for the host plant species of these weevils due to the relatively slow rate of molecular evolution of plant chloroplast DNA -39, whic species -43, havern Japan .C. sikkimensis living both in the broadleaved deciduous and evergreen forests and C. hilgendorfi living only in the broadleaved evergreen forests and in several plant species growing in these forests, shared a common phylogeographical pattern, with both exhibiting a genetic gap between the northeastern and southwestern parts of Japan. The geographic distributions of genetic diversity of plant species and those of their parasitic insects suggest that both were restricted to separate southwestern and northeastern refugia on the main islands of Japan during repeated glacial periods in the Quaternary. Moreover, these phytophagous insects, especially flightless or nonmigratory species, likely responded to the environmental changes associated with the glacial cycles in a fashion similar to that of their host plants. If the plant-insect association  remained stable throughout the Quaternary, the presence of the phytophagous insects may be an indicator of the coverage of the temperate forests. Thus, incorporating phylogeographical information about herbivorous insect species with that of their host plant species could more precisely elucidate the phylogeographical patterns of their associated forests.In this study C. sikkimensis among their associated host plant species, which suggests that weevils occupying different host species do not shape host races. Although the size and shape of the utilized acorns varied among plant species  similar past distributions of refugia between deciduous oak and evergreen forests, or (2) similar past host ranges. That is, C. sikkimensis might have been restricted to evergreen Castanopsis species in the last glacial age and may have recently expanded its host range independently in each refugium after the last glacial maximum. In the former case, if some populations of C. sikkimensis survived during the glacial periods within deciduous oak forests and others in evergreen forests in different isolated refugia, some host specialization could have arisen or have been reinforced. The observation that the weevils inhabiting different acorn species do not shape host races would suggest that the populations of C. sikkimensis inhabiting deciduous oak forests and those in evergreen forests were not isolated during the long glacial periods. At the last glacial maximum, global sea level is estimated to have dropped to approximately 140 m lower than the present level [C. sikkimensis expanded its host range to several deciduous and evergreen oak species independently from each refugium as recently as the last 20,000 years. In addition, in the European Curculio species, closely related weevils  do not associate with common host plant species [We did not find any genetic differentiation in the mtDNA of ies Fig. , C. sikknt level . The narnt level . This su species .C. sikkimensis and C. hilgendorfi strongly support the hypothesis that both deciduous oak and evergreen forest types survived together or adjacent to one another in small refugia during glacial ages, although these forests are presently distributed in cool and warm temperate zones in Japan, respectively. A more detailed investigation of the pollen records or further genetic studies of organisms living in these types of forests will provide further insight into the evolutionary history of the broadleaved deciduous and evergreen forests of Japan.The coincident patterns of C. sikkimensis in temperate forests of Japan. Comparing the phylogeographical patterns of two types of phytophagous insects  and of several plant species growing in these temperate zones of Japan enabled us to specify the historical processes of these forests more precisely. We demonstrated that during glacial ages the cool and warm temperate forests in Japan survived together, or adjacent to each other, at least in the southwestern and northeastern parts of the main islands. Our analyses provide a foundation for studying the evolutionary history of the broadleaved deciduous and evergreen forests in Japan, which characterize the biodiversity and endemism of the cool and warm temperate zones, where the effects of climate change are particularly severe.Our data suggest that geology and historical environment have contributed to shaping the recent genetic structure of KA designed of the study, performed the molecular genetic studies, and drafted the manuscript. MK and NM helped in drafting the manuscript. All authors have read and approved the final manuscript.


Aedes mosquitoes. After a quiescence of more than three decades, CF has recently re-emerged as a major public health problem of global scale. CF is characterized by an acute onset of high fever associated with a severe disabling arthritis often accompanied by prominent mucocutaneous manifestations. The disease is usually self-limiting, but the joint symptoms and some of the cutaneous features may persist after the defervescence. A wide range of mucocutaneous changes has been described to occur in association with CF during the current epidemic. Besides a morbilliform erythema, hyperpigmentation, xerosis, excoriated papules, aphthous-like ulcers, vesiculobullous and lichenoid eruptions, and exacerbation of pre-existing or quiescent dermatoses had been observed frequently. These unusual features may help in the clinical differential diagnosis of acute viral exanthems mimicking CF.Chikungunya fever (CF) is an arboviral acute febrile illness transmitted by the bite of infected  Aedes mosquitoes. Documented first time from an outbreak in Tanzania in 1952,[1Chikungunya fever (CF) is an acute viral illness caused by an arbovirus of the same name transmitted by the bites of in 1952,1 After anin 1952,1 The disein 1952,1A. albopictus, in addition to A. aegyptii, as an efficient transmitter of Chikungunya virus.[A human-mosquito-human cycle is responsible for the maintenance of the virus in the South-East Asia region in contrast to the sylvatic transmission cycle occurring in the African continent. The re-eya virus.CF may affect people of all age groups with an equal gender distribution. After an incubation period ranging from 3 to 12 days, there is usually an abrupt onset of high fever accompanied by severe arthralgias, myalgias, and skin rash. There may be conjunctival suffusion, persistent conjunctivitis, and cervical or sometimes generalized lymphadenopathy, together with swollen and tender joints frequently involving the small joints of the hand, wrist, and ankles, but also involving the larger joints such as knee and shoulder in some patients. Other clWhile the clinical feature of CF is dominated by the sudden onset of high fever and the disabling joint symptoms, mucocutaneous manifestations occur in a significant proportion of cases. A wide array skin and mucous membrane lesions have recently been documented in literature particularly by Indian workers looking specifically into this aspect of the disease during the current epidemic.–9 We havA large variety of skin and mucous membrane lesions have been documented to occur in association with CF including some that have not been described in other viral exanthems . The der106In a number of patients, hypermelanosis of the skin may develop soon after the rash has resolved . The hypAcute intertrigo-like lesions and peno-scrotal or perianal ulceration are other distinctive manifestations of CF. The patiVasculitic lesions and erythema nodosum like lesions have also been reported to occur in CF. Targetoi7Apart from the aphthous ulcerations and gingivitis, depigmenAlthough the acute febrile illness caused by the Chikungunya virus remits spontaneously without any sequelae in most patients, the joint manifestations may linger for a prolonged period of time. Persistent joint affection has been described to occur in about 12% of patients in the forms of residual stiffness without pain and persistent painful restriction of joint movements. NeurologAcute CF requires to be differentiated from a wide range of conditions that present with acute febrile exanthems with arthralgia. The principal Chikungunya mimic is dengue fever, which may occur as a co- infection in association with CF. It is ofDiagnosis of CF is based on a high index of suspicion while dealing with any case of acute febrile illness with joint symptoms particularly in the appropriate epidemiologic setting. While a morbilliform rash is common to many viral and bacterial exanthems, the unusual mucocutaneous features associated with CF, such as the rapidly developing facial hypermelanosis, aphthous-like ulcers, and the intense xerosis and scaling, may help in the differential diagnosis. A laboratory confirmation of diagnosis should ideally be done with the appropriate virological and serological tests, but these are rarely available in areas where the outbreak has occurred. Thus, the vast majority of cases are diagnosed on clinical and epidemiological grounds. A positive virus culture supplemented with neutralization provides a definitive proof for the presence of Chikungunya virus. A reversThere is no specific antiviral therapy available for CF. The disease is generally self-limiting and the goal of the therapy is symptomatic relief of complaints like fever and joint paint with paracetamol or NSAIDs. Since no vaccines are commercially available for inducing active immunity against the disease, the mainstays of prevention remain the vector control measures at the household and community levels and the avoidance of mosquito bites by appropriate measures.The mucocutaneous features are managed with symptomatic treatments like the use of oral antihistamines with or without application of soothing agents like calamine lotions for those patients who complain of pruritus associated with the rash. Xerosis and scaling may be treated with the application of mineral or vegetable oils or other emollients on moistened skin. We have seen facial hypermelanosis to persist for a long time after the remission of the acute febrile episode. This may be treated with topical hypopigmenting agents like hydroquinone with or without short-course topical steroids. Photoprotective measures including the usage of sunscreens should also be advised. Intertriginous and penoscrotal ulcerations may be treated with topical or systemic antibacterials for prevention or treatment of secondary bacterial infections.


This study aimed to investigate whether the use of female sex hormone drugs during pregnancy is a risk factor for subsequent breast and other oestrogen-dependent cancers among mothers and their children and for genital malformations in the children. A retrospective cohort of 2052 hormone-drug exposed mothers, 2038 control mothers and their 4130 infants was collected from maternity centres in Helsinki from 1954 to 1963. Cancer cases were searched for in national registers through record linkage. Exposures were examined by the type of the drug  and by timing . There were no statistically significant differences between the groups with regard to mothers' cancer, either in total or in specified hormone-dependent cancers. The total number of malformations recorded, as well as malformations of the genitals in male infants, were higher among exposed children. The number of cancers among the offspring was small and none of the differences between groups were statistically significant. The study supports the hypothesis that oestrogen or progestin drug therapy during pregnancy causes malformations among children who were exposed in utero but does not support the hypothesis that it causes cancer later in life in the mother; the power to study cancers in offspring, however, was very low. Non-existence of the risk, negative confounding, weak exposure or low study-power may explain the negative findings. © 1999 Cancer Research Campaign


To determine whether milk is able to transmit scrapie, 18 lambs with a prion protein genotype associated with high susceptibility to scrapie (VRQ/VRQ) were fed milk from twelve scrapie-affected ewes of the same genotype, and 15 VRQ/VRQ sheep reared on scrapie-free dams served as controls.The risk of scrapie infection increases with increased duration and proximity of contact between sheep at lambing. Scrapie infectivity has not been detected in milk but cellular prion protein, the precursor of disease-associated prion protein PrPd was detected in the distal ileum of the first two lambs, whilst PrPd was not found in lymphoreticular tissues in the third lamb. A control lamb, housed in a separate pen and culled at 38 days of age, was also negative for PrPd in a range of tissues. Samples of recto-anal mucosa associated lymphoid tissue collected from the remaining 15 live lambs at seven months of age (between five to seven months after mixing) were positive for PrPd in the scrapie milk recipients, whereas PrPd was not detected in the remaining 14 controls at that time. A subsequent sample collected from control lambs revealed PrPd accumulation in two of five lambs eight months after mixing with scrapie milk recipients suggestive of an early stage of infection via lateral transmission. By contrast, the control sheep housed in the same building but not mixed with the scrapie milk recipients were still negative for PrPd.Three lambs fed milk from scrapie-affected ewes were culled due to intercurrent diseases at 43, 44 and 105 days of age respectively, and PrPd in distal ileum and rectal mucosa indicates transmission of scrapie from ewe to lamb via milk (or colostrum) although it is not yet clear if such cases would go on to develop clinical disease. The high level of infection in scrapie-milk recipients revealed by rectal mucosal testing at approximately seven months of age may be enhanced or supplemented by intra-recipient infection as these lambs were mixed together after feeding with milk from scrapie-affected ewes and we also observed lateral transmission from these animals to lambs weaned from scrapie-free ewes.The presence of PrP The disease is usually confirmed by the detection of the disease-associated prion protein, PrPd is first detected in the lymphoid tissues of the digestive tract at two months of age [in utero or soon after birth. The oral route is consistent with an epidemiological study, which demonstrated that the risk of infection increased with increased duration and proximity of contact between sheep at lambing [d [d in prion disease, is found in the milk from ruminants therefore milk from an infected animal could serve as a source for PrPd and even transmit disease [Studies on the pathogenesis of natural scrapie in sheep have demonstrated that PrPs of age ,2, which lambing . The mai lambing  and PrPdmbing [d . Other pmbing [d . Parentembing [d ,7 or goambing [d  has not mbing [d . These f disease . In addi disease . The objFour lambs were culled as a result of intercurrent diseases: three lambs, two from a pair of lambs fed milk from different scrapie-affected ewes, were culled at 43 (lamb number 517/7), 44 (lamb number 516/7) and 105 days (lamb number 518/7) respectively. One building control lamb (number 548/7) was culled at 38 days of age.The remaining lambs .d in the lymphoid follicles  of two lambs (numbers 516/7 and 517/7) fed milk from scrapie-affected ewes revealed accumulation of PrPd was present in most or all of the lymphoid follicles of the examined section, with both tingible body macrophages (TBM) and moderate to marked follicular dendritic cell (FDC) immunolabelling  from the scrapie milk recipients, including the partner lambs of the lambs 516/7 and 517/7 which were culled early in the study, contained disease-associated PrP. PrPTable d-positive lambs that were culled with incidental diseases presented with PrPd-immunolabelling in the superficial inguinal lymph nodes. Lymphoid aggregates in the mammary gland were only visible in ewe 1010/6, and PrPd was present in three of six aggregates  that provided the milk for the PrPThe somatic cell count (SCC) in a milk sample collected each week from the ewes is listed in Table d is associated with early infection with scrapie [d in the distal ileum were infected with scrapie and that the source of infection was the milk from scrapie-affected ewes. Both ewes developed definite neurological signs of scrapie during the lactation. Immunohistochemical examination of RAMALT samples collected from the remaining recipient lambs at seven months of age showed PrPd accumulation in all lambs, including the partner lambs of the two culled, PrPd-positive animals. The absence of detectable PrPd in the distal ileum of the building control lamb, which was culled early in the experiment, and in the RAMALT samples from the remaining building controls collected twice within an interval of three months, supported our conclusion that environmental contamination was not responsible for infection with the scrapie agent. PrPd was also detected in the remaining live scrapie recipients at approximately seven months of age, at a median time of 199 days after mixing. The first RAMALT biopsy in the lateral transmission controls was taken at a median time of 162 days after introduction to the scrapie milk recipients and found no evidence of infection via lateral transmission at that time. The second sample collected at a median time of 242 days after mixing of controls with scrapie milk recipients  revealed that lateral transmission had already occurred in two of the five controls. This could imply that some of the scrapie milk recipients were in fact infected via lateral transmission from other PrPd-positive scrapie-milk recipients, such as the lambs culled early in the experiment, and not via milk from their corresponding scrapie-affected ewes. Considering the minimal amount of PrPd found in the distal ileum of the lambs culled early, it is unlikely that shedding of large amounts of PrPd occurred between partner lambs at mixing. Assuming the lateral transmission controls were exposed to a high dose of infectivity when first introduced to the scrapie milk recipients and that the infectivity continued to build-up in the intervening 150 days, the severity of labelling should have been at least similar to that of the scrapie milk recipients at their RAMALT examination. This was, however, not the case since three lambs had no detectable PrPd and two lambs were PrPd-positive, with TBM labelling only, and only up to half the follicles were positive. The accumulation pattern is similar for all gut-associated lymphoid tissue, including Peyer's patches and RAMALT, as well as lymph nodes: typically, TBMs are the first cells to become positive, followed by FDCs [d in any of the examined gut-associated lymphoid tissues of a culled scrapie milk recipient that was already mixed with others at eight days of age and culled at 105 days of age (lamb 518/7). Therefore the lamb with the weak labelling was probably infected via milk, but with a lower titre of infectivity.In naturally infected sheep of highly susceptible genotypes (including VRQ/VRQ), PrP scrapie ,2,12 and by FDCs . This su by FDCs . The dend in tonsils at approximately 19 months of age, although maternal transmission could not be completely excluded [d in two of five lateral transmission controls already at approximately eight months after mixing since they were housed in a previously scrapie-free environment and only had contact with the scrapie milk recipients, which were supposed to be in the early, pre-clinical stage of infection. Exposure to saliva , faeces or urine, which have shown no detectable scrapie infectivity in mouse or goat transmission studies [Lateral transmission of scrapie in the absence of ewes, which may shed the scrapie agent around parturition, has been demonstrated. VRQ/VRQ sheep, which had been born to TSE-free ewes introduced to a flock with a high incidence of scrapie resulting in potentially high environmental contamination, had detectable PrPexcluded . It was  studies , was mosd in mammary glands [d has not yet been detected in milk itself. Mastitis produces an increased SCC in milk and may result in milk carrying more infectivity, since it has been shown that TSE can be transmitted in sheep by transfusion of blood and blood cells [d in the lymphoid aggregates of the mammary gland of the ewe (1010/6) that provided the milk for one PrPd-positive lamb. Nine scrapie-affected ewes, including the only ewe (number 139/6) that fed a lamb that was without detectable PrPd in its lymphoreticular tissues, had somatic cell counts of above 100,000 cells/ml  at some number 19/6 that  in ewes -24. Indemia spp. . The SCCmia spp. . It is umia spp. . This wod was not detectable in the third culled lamb (number 518/7) fed milk from the scrapie-affected ewe 139/6. As mentioned earlier, involution of Peyer's patches and subsequent reduction of lymphoid follicles takes place from about 84 days of age in lambs [d-positive, culled lambs presented with only a few PrPd-positive follicles out of the total examined, it is possible that follicles with detectable PrPd may have been missed in this lamb. However, other lymphoid tissues, such as the mesenteric lymph nodes or the spleen, have been shown to contain PrPd from two months of age in naturally infected VRQ/VRQ sheep [d in the mesenteric lymph nodes and the spleen of the 105 day-old lamb thus suggests that the milk from the scrapie-affected ewe 139/6 did not carry enough infectivity to achieve PrPd accumulation in the lymphoid tissues, either because PrPd was not secreted via the milk at all or the amount of PrPd was not sufficient enough to accumulate in the lamb. The ewe's milk presented with a SCC as high as others but the length of lactation was shorter compared to most of the other ewes, although ewe number 140/6 also had a short lactation but fed a lamb that accumulated PrPd in RAMALT. However, this ewe's milk was discoloured and it is likely that blood cells rather than somatic cells present in the milk may have caused transmission. Unfortunately, the amount of collected milk in ewe 139/6 was too small to feed two lambs to confirm the absence of PrPd in RAMALT of a partner lamb.PrPin lambs , which m number 58/7 fed mRQ sheep . The absd is largely restricted to the gut-associated lymphatic tissue and nervous system, so prions may not be secreted in the milk. The lack of published information about infectivity of sheep milk has made it difficult to estimate the human health risk associated with milk from sheep and goats should BSE be found in small ruminants [We have demonstrated that scrapie can be transmitted to genetically susceptible lambs via milk. This model may or may not be applicable to scrapie in sheep of other genotypes, or to other species infected with other prion strains where the pathogenesis of prion infection may be different. For example, in scrapie cases of particular genotypes  and BSE uminants . It is iuminants , but lituminants  does howThese results indicate that there is a risk of the transmission of scrapie from ewe to lamb via milk or colostrum. Infection of lambs via milk may result in shedding of the infectious agent into the environment as we also observed infection of other lambs raised using scrapie-free ewes and then mixed in with the scrapie milk recipients. The study continues with the remaining lambs kept alive to assess if clinical disease develops, and further milk collection and feeding is planned to improve our estimate of the risk of transmission.All procedures involving animals were approved by the Home Office under the Animals (Scientific Procedures) Act 1986.136R154Q171 homozygotes as determined by genotyping the PrP gene at codons 136, 154 and 171 [Milk was collected by hand from twelve ewes  throughout their lactations. Weekly milk samples were collected after the first week of lactation , and the SCC was determined . Milk samples from individual ewes were stored at below -20°C for use in the following year. The ewes were kept in a flock with a high incidence of scrapie  and had  and 171 ). The TS and 171 , except  and 171 ,35 and iAll ewes were culled after developing definite clinical signs of scrapie, which were a combination of at least two of the signs 'changed behaviour (nervousness)', 'intense pruritus (rubbing)', 'wool loss or skin lesions', 'positive scratch test' , 'head tad libitum and a daily ration of concentrate feed. Mixing was conducted in stages due to the various amount of milk that was fed to the lambs , which is free from scrapie, and that were kept in a flock that had never had any cases of classical scrapie. This flock was established to provide TSE-free sheep for research projects and is maintained under strict biosecurity controls to prevent the occurrence of scrapie. The TSE-free status of the dams was confirmed by immunohistochemical examination of a section of the obex (see above) and Bio-Rad Platelia ELISA  on a proteinase-digested sample of the caudal medulla according to the manufacturer's instructions . Lambs w"Building controls" to control for environmental contamination comprised ten VRQ/VRQ Cheviot lambs that were born from ewes from the same NZ-derived flock (TSE-free status confirmed by post-mortem test as detailed above) and housed with their dams in the same building at around the same time as the recipient lambs but in separate pens. They were reared naturally on their dam and weaned at approximately eight weeks of age.It was acknowledged from the outset of the experiment that scrapie milk recipients had to be mixed because the number of available pens did not allow housing of small groups for a longer time period. In addition, sheep should be kept in groups to prevent distress caused by social isolation , and addWeaned control lambs received the same food as the weaned recipient lambs.d accumulation even in the very early stages of scrapie [Samples from lymphoreticular tissues  and t scrapie  and the A RAMALT sample was taken from each lamb at approximately seven months of age and examined by immunohistochemistry as described above. A second RAMALT biopsy was taken and examined similarly from all control lambs approximately three months later, which was equivalent to the maximum delay between mixing of scrapie milk recipients and introduction of lateral transmission controls.The images were captured using the microscope Eclipse E400 with attached digital camera DXM1200  and the image-processing and analysis software Lucia G, Version 4.82, . Photoshop Elements 5  was used for image cropping and text embedding.TK proposed the study with the help of SJB and HAS, wrote the manuscript and was the overall project manager. The projects supplying the sheep were managed by SJB and HAS. SJM performed the immunohistochemical examinations. All authors read and approved the final manuscript.


Some model experiments predict a large-scale substitution of Amazon forest by savannah-like vegetation by the end of the twenty-first century. Expanding global demands for biofuels and grains, positive feedbacks in the Amazon forest fire regime and drought may drive a faster process of forest degradation that could lead to a near-term forest dieback. Rising worldwide demands for biofuel and meat are creating powerful new incentives for agro-industrial expansion into Amazon forest regions. Forest fires, drought and logging increase susceptibility to further burning while deforestation and smoke can inhibit rainfall, exacerbating fire risk. If sea surface temperature anomalies (such as El Niño episodes) and associated Amazon droughts of the last decade continue into the future, approximately 55% of the forests of the Amazon will be cleared, logged, damaged by drought or burned over the next 20 years, emitting 15–26 Pg of carbon to the atmosphere. Several important trends could prevent a near-term dieback. As fire-sensitive investments accumulate in the landscape, property holders use less fire and invest more in fire control. Commodity markets are demanding higher environmental performance from farmers and cattle ranchers. Protected areas have been established in the pathway of expanding agricultural frontiers. Finally, emerging carbon market incentives for reductions in deforestation could support these trends. The future of the Amazon Basin is a topic of great concern worldwide. The trees of the Amazon contain 90–140 billion tons of carbon . The remet al. regional climate change on the Amazon fire regime. Could accelerating, forest-substituting and forest-damaging economic activities interact with regional climate change to replace or degrade a large portion of the Amazon forest over the next two decades? And what counteracting trends could prevent such a dieback? These questions are the focus of this paper. We review current trends in Amazon economic, ecological and climatic processes, the growing evidence of positive feedbacks among these processes and the potential for these interactions to push Amazon forests towards a tipping point. We conclude with a brief review of some of the processes that could contribute to a strategy to reduce the chances of a major Amazon forest dieback. This synthesis draws on the published literature, some unpublished data, and presents two new maps of the Amazon.These ecological services may be threatened by global warming through a late-century, climate-driven substitution of forests by savannah and semi-arid vegetation in what has been called the Amazon forest ‘die back’ et al. . HoweverCerrado and Amazon regions  may increase the prospect of further disturbance from fire for years afterwards , grasses , bamboo (Guandu spp.) and ferns  now dominates soils once covered by moist tropical forest across approximately 300 000 km2  high levels of tree mortality are induced by drought, fire, fragmentation or logging, (ii) propagules of high-fuel grasses and, perhaps, ferns or bamboo are abundant following tree mortality, (iii) ignition sources are present, and (iv) the forest is subjected to severe seasonal or episodic drought. The portion of the Amazon under which these conditions are all met may be expanding.et al. et al. Several lines of evidence suggest that the eastern Amazon may become drier in the future, and that this drying could be exacerbated by positive feedbacks with the vegetation. At the broadest temporal and spatial scale, most global circulation models (GCMs) predict that greenhouse gas accumulation and associated increases in the radiative forcing of the atmosphere will cause a substantial (more than 20%) decline in rainfall in eastern Amazonia by the end of the century, with the biggest decline occurring in the dry season et al. . The lowA more likely near-term shift in Amazon climate may be associated with changes in sea surface temperature that are usually associated with Amazon drought. Rainfall tends to decline in the Amazon when sea surface temperatures rise along the Pacific coast of northern South America through El Niño episodes , much as they did in 1998 and 2005 . This 2-year moratorium on the purchase of soya planted on soils recently cleared from Amazon forest was stimulated by a campaign against Amazon soya initiated by the environmental organization Greenpeace . Soya pret al. 2007, personal communication), and will be favoured if land prices escalate through growing demands for Amazon soil.Third is the prospect of restricting the advance of Amazon cattle ranching. Approximately one-quarter of the previously forested lands in the Brazilian Amazon are in some stage of abandonment . One wayThe prospect of restricting the expansion of agricultural and livestock production in the Amazon to those lands of high suitability becomes plausible in the light of a fourth important trend. Most parks and biological reserves in the Amazon have been created far from the agricultural frontier, where the opportunity costs of conservation are low and where, unfortunately, these protected areas exert little influence on deforestation . This hiA fifth potentially robust mechanism for counteracting the pressures to clear and degrade Amazon forest, and that could reinforce efforts to constrain the expansion of cattle ranching, is emerging within the United Nations Framework Convention on Climate Change (UNFCCC) negotiations. The proposal to compensate those tropical nations that succeed in reducing their nationwide emissions of greenhouse gases from deforestation , such a Synergistic trends in Amazon economies, forests and climate could lead to the replacement or severe degradation of more than half of the closed-canopy forests of the Amazon Basin by the year 2030, even without invoking fire or global warming. Counteracting these trends are emerging changes in landholder behaviour, recent successes in establishing large blocks of protected areas in active agricultural frontiers and practical techniques for concentrating livestock production on smaller areas of land that could reduce the likelihood of a large-scale self-reinforcing replacement of forest by fire-prone brush. In the long term, however, the avoidance of an Amazon forest dieback may depend upon worldwide reductions of greenhouse gas emissions that are large enough to prevent global temperatures from rising more than a degree or two.


Neoceratodus forsteri, which is the most primitive of all the lungfish and possibly the closest living relative to the ancestors of tetrapods.The transition from water to land was a key event in the evolution of vertebrates that occurred over a period of 15–20 million years towards the end of the Devonian. Tetrapods, including all land-living vertebrates, are thought to have evolved from lobe-finned (sarcopterygian) fish that developed adaptations for an amphibious existence. However, while many of the biomechanical and physiological modifications necessary to achieve this feat have been studied in detail, little is known about the sensory adaptations accompanying this transition. In this study, we investigated the visual system and visual ecology of the Australian lungfish Neoceratodus have five spectrally distinct retinal visual pigments. A single type of rod photoreceptor contains a visual pigment with a wavelength of maximum absorbance (λmax) at 540 nm. Four spectrally distinct single cone photoreceptors contain visual pigments with λmax at 366 (UVS), 479 (SWS), 558 (MWS) and 623 nm (LWS). No double cones were found. Adult lungfish do not possess UVS cones and, unlike juveniles, have ocular media that prevent ultraviolet light from reaching the retina. Yellow ellipsoidal/paraboloidal pigments in the MWS cones and red oil droplets in the LWS cones narrow the spectral sensitivity functions of these photoreceptors and shift their peak sensitivity to 584 nm and 656 nm, respectively. Modelling of the effects of these intracellular spectral filters on the photoreceptor colour space of Neoceratodus suggests that they enhance their ability to discriminate objects, such as plants and other lungfishes, on the basis of colour.Juvenile The presence of a complex colour vision system based on multiple cone types and intracellular spectral filters in lungfishes suggests that many of the ocular characteristics seen in terrestrial or secondarily aquatic vertebrates, such as birds and turtles, may have evolved in shallow water prior to the transition onto land. Moreover, the benefits of spectral filters for colour discrimination apply equally to purely aquatic species as well as semi-aquatic and terrestrial animals. The visual system of the Australian lungfish resembles that of terrestrial vertebrates far more closely than that of other sarcopterygian fish. This supports the idea that lungfishes, and not the coelacanth, are the closest living relatives of the ancestors of tetrapods. Lungfishes (Dipnoi) diverged from the main vertebrate stock in the early Devonian ca. 390 MYA)  and toge90 MYA  aProtopterus sp.) and South American (Lepidosiren) lungfishes belong to the Lepidosirenidae; the Australian lungfish  of the retinal photoreceptors, the spectral transmittance of the ocular media, the spectral distribution of the ambient light in a typical lungfish habitat and the spectral reflectance of relevant objects in its environment. Using these data, we have modelled the performance of the lungfish visual system with respect to its ability to distinguish both real and theoretical coloured objects.Thus, the polychromatic visual system of the lungfish is at odds with its known behavioural characteristics and raises a number of interesting questions with regard to the neuroethology of the lungfish, the function and utility of coloured oil droplets and the evolution of the tetrapod visual system. To investigate these issues, we have measured some of the most important parameters of the visual ecology of max 366 nm), short- , medium-  and long-wavelengths . We did not observe double cones in the lungfish retina, a finding that supports previous anatomical studies . Similane types .Fundulus heteroclitus , only sourless" , althougspectrum . In factspectrum  and fresspectrum , the facspectrum  suggestsThe red oil droplets of lungfish LWS cone photoreceptors resemble closely those of some terrestrial vertebrates and, therefore, provide circumstantial support for the findings from molecular genetic studies that lungfishes are more closely related to the ancestors of the tetrapods than other extant sarcopterygian fish [e.g. ], which There may be other reasons for having yellow and red spectral filters besides colour vision. These include the absorption of scattered short-wavelength light, which would improve visual contrast detection by the MWS and LWS single cones in the same way that UV-blocking 'haze' or yellow filters improve contrast in photography . UltraviVallisneria spiralis [Daphnia or other planktonic prey; zooplankton often absorb or scatter UV wavelengths differently to the surrounding water and this enhancement in visual contrast may be used by predators for initial detection of, or discrimination between, preferred prey species [UVS cones were not found in the retina of the adult lungfish and, although they may have been present in such low numbers that they were overlooked during the microspectrophotometric study, the greatly increased absorption of short wavelengths by the ocular media of adults, compared to those of juveniles Fig. , suggestspiralis . UV sens species ,40. Howe species . MoreovePerca flavescens) have small single cones containing a 400 nm λmax visual pigment that are lost entirely from the retinal mosaic as the fish matures and becomes demersal [Salmo trutta) and rainbow trout  also lose their UVS cones (λmax 340–360 nm) during development [Neoceratodus [The apparent loss of UVS cones from the adult lungfish retina reflects the situation observed in some teleost fishes that also show ontogenetic shifts in preferred habitat. For example, surface-feeding juvenile yellow perch  protein when conjugated with 11-cis retinal, and extends the extreme long wavelength limit of vertebrate visual pigment λmax  from about 575 to 625 nm [max by the yellow ellipsoidal/paraboloidal pigmentation, the enhanced long-wavelength sensitivity of Neoceratodus suggests that the discrimination of objects differing in their reflectance at long wavelengths is very important. In this regard, it is interesting to note that many of the macrophytes, including those  that lungfish use for food, shelter or spawning [At the other end of the spectrum, the peak sensitivity of the LWS cone (656 nm) in adult ronments . Habitatronments , lampreyronments ,46 and ao 625 nm ,48. MoreNeoceratodus is also shifted considerably towards longer wavelengths, having a visual pigment with a λmax at 540 nm. This spectral shift is mostly the result of the use of 3,4-didehydroretinal rather than 11-cis retinal as the chromophore and is similar to the situation observed in the 3,4-didehydroretinal-based rod visual pigments of teleost fish such as the rudd Scardinius erythrophthalmus (λmax 540 nm) [Perca fluviatilis (λmax 540 nm) [Neoceratodus, these fishes often inhabit green fresh water bodies that are relatively richer in long wavelengths of light.The spectral sensitivity of the rod photoreceptor of  540 nm)  and the  540 nm) . Like NeNeoceratodus may also play a role in the assessment of body colouration in conspecifics. Neoceratodus are strongly countershaded  or 4 (juveniles) cone photoreceptor types. Some of the cones possess coloured intracellular filters that tune the spectral sensitivity functions of the photoreceptors, enhancing the ability to discriminate between objects on the basis of colour, including those of ecological significance. In this respect, the visual system of the lungfish is much more like that of a turtle or a bird than an amphibian or a fish. The close phylogenetic relationship of the lungfish to the ancestors of the tetrapods, suggests that many of the ocular characteristics seen in extant terrestrial vertebrates may have evolved in water prior to the transition onto land.-1) and euthanased by spinal cord transection followed by pithing. All procedures were approved by the University of Queensland Animal Ethics Committee.Two adult lungfish  were caught from the Enogerra Reservoir, Queensland, Australia. Three captive-bred juvenile lungfish  were donated by Prof. Jean Joss of Macquarie University, Australia. Animals were kept in large freshwater aquaria at the University of Queensland under a 12:12 light:dark cycle and fed a diet of commercial fish food. Animals were dark-adapted overnight, anaesthetised with benzocaine  were dissected away and mounted on a 24 × 60 mm No. 1 glass coverslip in a drop of physiological saline containing 10% dextran . The retina was gently teased apart using mounted needles and the preparation covered with a 22 × 22 mm No. 0 glass coverslip. The edges of the top coverslip were sealed with nail varnish to prevent dehydration and movement of the retinal tissue.Eyes were removed under dim red light and all subsequent procedures were conducted under infra-red illumination with the aid of a dissecting microscope fitted with an infra-red image converter . The retina was removed from the eye and placed in physiological saline . Small pTransverse absorption spectra (330–800 nm) of the inner and outer segments of retinal photoreceptors were made using a single-beam, wavelength-scanning, computer-controlled microspectrophotometer described in detail elsewhere ,52. Briein toto. In each case, light from an Ocean Optics PX-2 Pulse Xenon Lamp  was delivered to the rear of the eye/lens/cornea using a 1000 μm diameter quartz fibre optic and transmitted light collected by a 50 μm diameter quartz fibre optic connected to an Ocean Optics S2000 spectroradiometer [The spectral transmittance (300–900 nm) of the ocular media was measured in eyes taken from one of the juvenile and one of the adult lungfish used for microspectrophotometric analysis. In the adult, the cornea and lens were measured separately and the transmittance spectra combined. In the juvenile, a portion of the sclera at the back of the eye directly opposite the pupil was removed and the ocular media (including aqueous and vitreous humours) measured diometer . TransmiNeoceratodus habitat: at the entrance to Chinaman's Creek in the Mary River, near Tiaro, Queensland, at midday on the 3rd October 2003 (near the end of the dry season). Measurements were made 2 m out from the riverbank with readings taken at the surface and at various depths up to 1.25 m, as described elsewhere [Downwelling irradiance spectra (300–800 nm) were measured using an Ocean Optics S2000 spectroradiometer in a typical lsewhere ,59. WateNeoceratodus were made using an S2000 spectrometer, as described previously [Vallisneria spiralis, Nitella sp., Ceratophyllum demersum and Elodea canadensis taken from the Brisbane River at College's Crossing, Ipswich, Queensland, where lungfish are known to spawn . Reflectance measurements were also made of the scales on the dorsal surface and two different areas of the ventral surface (one pink and one orange to the human eye) of an adult lungfish (TL 110 cm). All reflectance spectra were measured relative to a white standard  and were smoothed with an 11-nm unweighted running average.Spectral reflectance measurements (300–800 nm) of objects of potential biological relevance to eviously . Measure-1. Spectral sensitivity calculations were also made for the theoretical case where coloured filters are lacking from the cone inner segments, for comparison in the modelling of visual performance described below.The quantal spectral sensitivity of each cone type in both juvenile and adult lungfish was calculated as the product of outer segment axial absorptance, oil droplet or ellipsoidal/paraboloidal pigment axial transmittance and ocular media transmittance . The phon-dimensional colour space, where n is the number of photoreceptor types involved in colour vision. All theoretically possible surface colours occupy a part of that colour space, and together form a conceptual shape known as an object-colour solid. Each colour locus within the colour space is surrounded by an elementary volume containing very similar colours from which it is indistinguishable. The number of colours that can be discriminated by a given visual system is defined as the total number of these elementary volumes contained within the object-colour solid.To determine the theoretical visual benefit of spectral filters  within the inner segment of lungfish photoreceptors, we first used the 'object-colour solid' method of Vorobyev ,25. Brier) is given as r = b/d, where b is the (gross) benefit due to the change in shape of the object-colour solid and d is the factor describing the cost of the spectral filters due to the increased noise resulting from reduced transmission of photons to the visual pigment; b = G/G0, where G and G0 denote the shape factor of the object colour solid with and without oil droplets, respectively [Neoceratodus is not known we do not present the data for bright light illumination. However, for the limiting case of dim light illumination, both the cost and benefit of coloured spectral filters can be calculated using microspectrophotometric data alone [When fluctuations in the number of quanta (photons) absorbed by the cones determines colour discrimination thresholds, this elementary volume is proportional to the product of the square roots of the number of quanta absorbed by each receptor channel per integration time and per receptive field . This alectively . In brigectively . The benectively ; becauseta alone .Neoceratodus to investigate the appearance of real, behaviourally relevant objects found in their environment. While the object-colour solid method allows us to draw conclusions based on all possible colours and examine the ultimate limits of the lungfish colour vision system, receptor space modelling allows us to address more ecologically driven questions, such as the ability to discriminate between different types of vegetation and detect subtle differences in conspecific body colouration.We also modelled the cone photoreceptor 'colour space' of n receptors is represented as an n-dimensional receptor space, the co-ordinate axes of which correspond to the relative photon catches of each cone photoreceptor type. Assuming that the colour-opponent mechanisms responsible for colour discrimination in the lungfish retina signal only chromatic (colour) differences between an object and the background against which it is viewed , achromatic (intensity) information can be disregarded [n-1 dimensions. In such a plot, pure excitation of any one cone type is represented by a locus at the relevant vertex; colours that excite more than one cone type are plotted according to their relative excitation ratios. The end result is a series of points whose relative distance from each other provides an indication of the likely discriminability of the colours to the visual system under examination, based on hue alone.The calculation of receptor spaces (and their associated chromaticity diagrams) is described in detail by Kelber et al. . Brieflyregarded . ReceptoReflectance spectra of lungfish colours and other relevant objects, measured relative to a white standard as described above, were multiplied by the downwelling irradiance spectrum (habitat light) and then multiplied by the quantal spectral sensitivity of each cone type. The resultant photon catch for each cone type was then divided by the sum of the photon catches for all cone types (to give relative photon catch) and plotted on the chromaticity diagram. The modelling was performed for both the juvenile and adult lungfish visual system, with and without the filtering effect of the coloured intracellular filters taken into account. Because there are no measurements of photoreceptor channel noise (Weber fractions) in lungfishes, we were unable to quantify the physiological relevance of any changes in colour distances.NSH carried out the microspectrophotometric measurements; assisted with animal capture, habitat light spectra measurements and colour space modelling; and wrote the manuscript. HJB coordinated animal capture and made the measurements of ocular media transmittance, spectral reflectance of lungfish and other object colours and habitat light spectra with NJM. MV and NJM assisted with colour space modelling. SPC helped with the conception of the project and assistance in the field. All authors read, contributed to and approved the final manuscript.


Several studies have suggested that the specific stroke subtype may influence the presence of leukoaraiosis in patients with ischemic stroke. We investigated the association between stroke subtype and leukoaraiosis in Korean patients with ischemic stroke by MRI.There were 594 patients included in this study that were classified as large artery disease, lacune and cardioembolic stroke. For large-artery disease, the analysis focused on the intracranial or extracranial location of the stenosis, and the multiplicity of the stenotic lesions. Leukoaraiosis grading was performed according to the Atherosclerosis Risk in Communities Study.There was a significant association between leukoaraiosis and the stroke subtypes; the large-artery-disease group had a higher prevalence of leukoaraiosis than did the other groups . On the multivariate linear regression analysis, age, the presence of hypertension, previous stroke and stroke subtype were independently associated with the presence of leukoaraiosis. In the sub analysis of the large-artery-disease group, the leukoaraiosis had a tendency to be more prevalent in the mixed and intracranial stenosis group than did the extracranial stenosis group .The association of leukoaraiosis with large-artery disease in this study might be due to the relatively high prevalence of intracranial occlusive lesions in Korean stroke patients compared to other ethnic groups. The term leukoaraiosis (LA) refers to lesions of altered signal intensity on computed tomography (CT) and magnetic resonance imaging (MRI) in the periventricular and subcortical white matter. LA is found during the normal aging process, and in the patients with cerebrovascular disease. It also constitutes the core pathology of Binswanger's disease, a type of vascular dementia. The association of LA with lacunar infarcts rather than territorial infarcts is well documented -7. HowevIn the present study, we analyzed the association between stroke subtype and LA in Korean stroke patients using MRI.We initially included 963 consecutive acute ischemic stroke patients admitted to the neurology department from July 2003 to June 2007. All patients underwent detailed clinical evaluation including laboratory tests, chest radiography, transcranial Doppler study, electrocardiography and 24 hour Holter monitoring. In addition, transthoracic echocardiography and brain magnetic resonance imaging (MRI), contrast-enhanced MR angiography (MRA) and/or cerebral angiography were obtained. All results from the evaluations were analyzed according to the diagnostic criteria for stroke mechanisms and etiology based on the TOAST subtype classification system . Among tFinally, 594 cases with large artery disease (297 patients), lacune (193 patients) and cardioembolic stroke (104 patients) were enrolled in this study. The ethics committee at our institution approved the study protocol, and all subjects provided written informed consent.The clinical information included age, gender, history of hypertension (defined by the use of an antihypertensive agent before admission or a systolic pressure > 140 mmHg or diastolic pressure > 90 mmHg demonstrated on repeated examinations at least one month after presentation with a stroke), diabetes mellitus (defined as a fasting blood glucose level > 126 mg/dl or a history of being treated for diabetes mellitus) and hyperlipidemia . In addition, regular cigarette smoking, a previous history of ischemic stroke and heart disease  were notedAll patients enrolled underwent conventional MRI on a 1.5-T system  within 7 days of the stroke onset. The conventional MRI consisted of transverse T2/T1-weighted, fluid-attenuated inversion recovery (FLAIR) sequences and sagittal T1 with 5-mm-thick slices. Diffusion-weighted imaging was obtained in the transeverse plane using a single-shot echoplanar, spin-echo pulse sequence. A three-dimensional time of flight MRA of the intracranial arteries and contrast-enhanced MRA of the head and neck were also performed on the same system using a neurovascular coil.LA was defined as a periventricular white matter lesion with hyperintensity on T2- weighted and FLAIR images and without prominent hypointensity on T1-weighted images. The LA grading was according to the Atherosclerosis Risk in Communities (ARIC) study ,10. ThreThe evaluation of the arterial stenosis was performed by the investigators and > 50% of signal loss on the MRA was considered to be a "significant stenosis" for the classification of stroke subtype and the categorization of stenosis pattern. The locations of significant stenosis were categorized as located in the intracranial or extracranial arteries. For the internal carotid artery, an intracranial location was defined when the stenotic lesion was distal to the ophthalmic artery. For the vertebral artery, the differentiation was made at the point where the artery pierced the dura at the level of the foramen magnum. The intracranial extent of the stenosis included up to the M2 of the middle cerebral artery (insular segment which terminates at the circular sulcus of the insula before the operculum) and the A2 segment of the anterior cerebral artery (ascending segment with inferior forward convexity) in the anterior circulation, and the P2 segment of the posterior cerebral artery . The area of the stenotic lesion was divided into intracranial or extracranial and anterior or posterior circulation. The stenoses were described as single or multiple according to the number of the areas involved with the arterial stenosis.According to the distribution and pattern of the stenotic lesions, the patients with large-artery disease were categorized as intracranial, extracranial and a mixed  group, and as multiple- or single-stenosis groups.We classified persons with leukoaraiosis of grade 3 or higher as having "LA" and of grade 2 or lower as having "little or no LA" . The preOf the 594 patients, 342 (57.6%) were men and 252 (42.4%) were women . In study population, distinct white matter changes were present in 307 patients (51.7%). The association of the presence of LA to age was statistically significant; the LA group had higher age at onset of stroke than the "little or no LA" group. The LA was more frequently observed in female gender, the patients with hypertension and a history of previous ischemic stroke than in the patients without this history (P < 0.05). There was no significant association between the LA and diabetes mellitus or hyperlipidemia; a negative correlation was found with the smoking status and the presence of heart disease  were in the large-artery-disease group, 93 patients (30.3%) in the lacunar group and 44 patients (14.3%) in the cardioembolic group ; extracranial lesions were uncommon. The LA tended to be more frequent in patients with intracranial stenoses than in the patients with extracranial stenoses alone. The intracranial location for cerebrovascular atherosclerosis is characteristic of strokes in the Asian population -31. TherUnlike extracranial stenoses, more prevalent in Caucasians, intracranial stenoses may have a different pathogenesis contributing to the development and progression of LA. First, atherosclerotic stenoses of intracranial arteries can directly occlude the orifice of numerous small perforators penetrating into the deep brain parenchyma. The occlusion of the small perforators promotes extensive hypoperfusion of the periventricular region that is vulnerable to ischemia. Second, the periventricular border zone, under ischemic conditions induced by the stenoses of intracranial arteries, might have less opportunity to be compensated by blood flow via major collateral channels such as the anterior or posterior communicating artery, which can be recruited without difficulty in stroke patients with stenosis of extracranial vessels. Third, compared with the emboli from the stenotic lesions of extracranial arteries, those from an intracranial location might not easily be cleared away by travel along with the blood flow, probably because of the close proximity to the perfused brain tissues.The limitations of this study include the following. One methodological problem is that our results are based on a cross-sectional sample. The longitudinal effect of large-artery atherosclerosis on periventricular white matter changes could not be accurately assessed in this study. In addition, a selection bias might have been present because 369 38.3%) of the 963 patients were excluded due to the diagnosis of a stroke of undetermined etiology or other determined etiology; we used very strict criteria for the classification of subjects into specific stoke subtypes. For example, suspected large artery disease with < 50% signal loss of the proximal artery on MRA, or supratentorial subcortical infarcts of < 1.5 cm with > 50% stenosis of the parent artery were classified as strokes of undetermined origin. The application of this criterion would increase the specificity and lessen the likelihood of misclassification of patients in the other categories [.3% of thThe results of this study showed that LA is significantly associated with large-artery-disease rather than other stroke subtypes including small vessel disease in Korean stroke patients. The differences between our study and previous reports might be due to the high prevalence of intracranial occlusive lesions in patients with cerebrovascular atherosclerosis in the Asian population. Different from the high-grade stenoses of extracranial arteries, the stenoses of intracranial arteries might induce extensive white matter changes by interrupting blood flow to the periventricular border zone vulnerable to ischemia.The authors declare that they have no competing interests.SJL and JSK conceived and coordinated the study, analysed the data and drafted the initial manuscript. All authors were involved in initial literature search and collection of data. Review of initial manuscript for major intellectual content was done by SJL and JSK. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The prokaryotic toxin-antitoxin systems  are widespread, mobile two-gene modules that can be viewed as selfish genetic elements because they evolved mechanisms to become addictive for replicons and cells in which they reside, but also possess "normal" cellular functions in various forms of stress response and management of prokaryotic population. Several distinct TAS of type 1, where the toxin is a protein and the antitoxin is an antisense RNA, and numerous, unrelated TAS of type 2, in which both the toxin and the antitoxin are proteins, have been experimentally characterized, and it is suspected that many more remain to be identified.We report a comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems in prokaryotes. Using sensitive methods for distant sequence similarity search, genome context analysis and a new approach for the identification of mobile two-component systems, we identified numerous, previously unnoticed protein families that are homologous to toxins and antitoxins of known type 2 TAS. In addition, we predict 12 new families of toxins and 13 families of antitoxins, and also, predict a TAS or TAS-like activity for several gene modules that were not previously suspected to function in that capacity. In particular, we present indications that the two-gene module that encodes a minimal nucleotidyl transferase and the accompanying HEPN protein, and is extremely abundant in many archaea and bacteria, especially, thermophiles might comprise a novel TAS. We present a survey of previously known and newly predicted TAS in 750 complete genomes of archaea and bacteria, quantitatively demonstrate the exceptional mobility of the TAS, and explore the network of toxin-antitoxin pairings that combines plasticity with selectivity.The defining properties of the TAS, namely, the typically small size of the toxin and antitoxin genes, fast evolution, and extensive horizontal mobility, make the task of comprehensive identification of these systems particularly challenging. However, these same properties can be exploited to develop context-based computational approaches which, combined with exhaustive analysis of subtle sequence similarities were employed in this work to substantially expand the current collection of TAS by predicting both previously unnoticed, derived versions of known toxins and antitoxins, and putative novel TAS-like systems. In a broader context, the TAS belong to the resistome domain of the prokaryotic mobilome which includes partially selfish, addictive gene cassettes involved in various aspects of stress response and organized under the same general principles as the TAS. The "selfish altruism", or "responsible selfishness", of TAS-like systems appears to be a defining feature of the resistome and an important characteristic of the entire prokaryotic pan-genome given that in the prokaryotic world the mobilome and the "stable" chromosomes form a dynamic continuum.This paper was reviewed by Kenn Gerdes (nominated by Arcady Mushegian), Daniel Haft, Arcady Mushegian, and Andrei Osterman. For full reviews, go to the Reviewers' Reports section. Bacterial toxin-antitoxin systems  originally have been characterized in the 1980s as molecular systems encoded in plasmids and ensuring the persistence of a plasmid in a host lineage during replication by making the cells "addicted" to the plasmid so that only plasmid-containing daughter bacteria survived after a cell division . As implThe TAS are currently classified into two major types on the basis of the nature of the antitoxin ,6. Type Type II TAS employ a protein antitoxin to keep the toxin inactivated via protein-protein interaction. These systems show considerable structural and functional diversity among both the toxins and the antitoxins. The most common activity of Type II toxins seems to be that of an mRNA-specific endonuclease, termed interferases ,9. In parelBE operon. The end result is the extensive cleavage of ribosome-associated mRNA, a major shutdown of translation, and concomitant increase of the pool of charged tRNAs , the boundary between selfishness and "normal" cellular function seems to be fuzzy and more a matter of convention than a real distinction."If this is so, what is a reason to distinguish resistome from the rest of the mobilome? After all, any biosynthetic operon that is mobile may be "selfish" in the sense of J. Lawrence , but it also has "normal" cellular function. Moreover, the function of a considerable portion of TAS is not (or not only) resistance.Authors' response: Of course, these boundaries are intrinsically fuzzy. Nevertheless, we believe that the components of the resistome do show a characteristic balance between selfishness and "normal" functioning that distinguishes them, on one end of the mobility range, from transposons and other "genuine" mobile elements, and on the other end, from regular biosynthetic operons. The latter, probably, should not be considered bona fide members of the mobilome although any operon indeed shows a degree of selfishness sensu Lawrence, so that there is not sharp boundary between the mobilome and the rest of the prokaryotic gene pool as noted here and elsewhere . ObjectTwo general questions:A. Per the "compromise" proposal and other observations: does it follow that antitoxin genes may be distributed broader than the toxin genes ? Is this actually observed, or is there perhaps a wash with some antitoxins represented by small RNAs?"A potential compromise between a purely selfish life style of TAS and integral cellular functions could be a role of chromosomally encoded TAS in the protection of prokaryotic cells against post-segregational killing induced by plasmid-encoded homologous TAS whereby the antitoxin encoded by a chromosomal gene sequesters a plasmid-encoded toxin. Experimental evidence of such protection was reported, and elimination of the chromosomal TAS in the presence of the respective plasmid did adversely affect the fitness of the host bacterium ."Authors' response: We do find many more antitoxins than toxins , an indeed some TAS are encoded by phage genomes.What is the connection of TAS to CRISPR loci? Are there any short toxin-derived sequences in CRISPRs?Authors' response: There is no particularly strong link. Some TAS occur in the vicinity of CRISPR loci but so do many other genes that might belong to the broadly defined mobilome.Is there any correlation between the number of both types of loci in different genomes?Authors' response: No correlation beyond what is expected from the scaling of genes in a particular category with the genome size.Minor comments:"Resistome domain of the prokaryotic mobilome": do we really speak like this?Authors' response: yes, we (the authors) see nothing wrong about it and so do speak like this. The reader is informed by this comment that others do not."The "selfish altruism", or "responsible selfishness", of TAS-like systems..." Is this Chernyshevsky-Randian property of TAS-like systems a defining feature of the whole mobilome, or of its subset resistome?Authors' response: We appreciate this point and the fully appropriate references to Chernyshevsky  and Ayn Rand. Upon consideration, we indeed replaced "mobilome" with "resistome" as some other members of the mobilome might show less restrain and responsibility.The first sentence of Conclusions. Replace "We report here the most detailed and comprehensive comparative-genomic analysis of type II TAS so far available (to our knowledge)" by "We report here a comparative-genomic analysis of type II TAS"?Authors' response: We understand that this could read like honking our horn. However, the phrase carries a message. We kept it.Andrei Osterman, The Burnham InstituteThe excellent article by Makarova, Wolf and Koonin could be indeed termed Genomic Encyclopedia of Prokaryotic TAS-2 for the breadth and depth of coverage of this fascinating system over 750 prokaryotic genomes. However, the accurate cross-genomic projection of the presently known TAS components, no matter how challenging and important, is only one of the remarkable accomplishments of this study. By pushing the boundaries of predictive comparative genomics to a completely new level, the authors also substantially expanded a repertoire of known toxins and antitoxins. They developed a sophisticated workflow elegantly capturing the elusive and mysterious nature of TAS operons, which allowed them to predict dozens of previously unknown players and combinations thereof. This workflow  is yet another remarkable deliverable of this study, which will be inspirational and instructive for those who are chasing after other challenging protein families. The detailed analysis of the resulting monumental picture, a genomic distribution of thousands of TAS elements, provided new insights into their evolution, intra- and interspecies mobility and functional associations. No doubt, this single study provided a great starting point for dozens of experimental biologists to follow upon.The list of 315 adjacent pairs of COGs. This file contains the list of 315 adjacent pairs of COGs that were further analyzed on a case by case basis for prediction of TAS (approach 1) as described in the Methods section.Click here for fileMultiple alignment of representative sequences of COG5606. The alignment supports the analysis and description of the predicted RelE-COG5606 TASClick here for fileMultiple alignment of truncated members of the MerR-like family. The alignment supports the analysis and description of the predicted PIN-MerR TASClick here for fileMultiple alignment of representative sequences of the XF1663 family. The alignment supports the analysis and description of the predicted MazF-XF1663 TASClick here for fileMultiple alignment of representative sequences of the YhfG family. The alignment supports the analysis and description of the predicted YhfG-Fic/Doc TASClick here for fileMultiple alignment of representative sequences of the DUF397 family. The alignment supports the analysis and description of the predicted DUF397-HTH TASClick here for fileMultiple alignment of representative sequences of the YgiU family. The alignment supports the analysis and description of the predicted YgiU-xre TASClick here for fileMultiple alignment of representative sequences of COG3832. The alignment supports the analysis and description of the predicted COG3832-ArsR TASClick here for fileMultiple alignment of a representative set of GNAT family acetyltransferases. The alignment supports the analysis and description of the GNAT-xre and GNAT-RHH systemsClick here for fileThe representation of toxins, antitoxins and toxin-antitoxin pairs in 750 complete genomes of prokaryotes. This file contains the counts of solo toxin and antitoxin genes as well as TA-pairs for all 750 prokaryotic genomes analyzed in this work. Phenotypic characteristics and the total number of protein coding genes are given for each genome.Click here for fileStatistical Appendix for the Methods. The appendix contains additional details concerning the following procedures: i) the likelihood model used to estimate the scaling parameters for the number of TAS pairs relative to the genome size and ii) variability of abundance of different TAS pairs within ATGCs.Click here for fileTable S1. Distribution of TAS among prokaryotic taxaClick here for fileSpecies abbreviations used in the alignments in Figures and 6Click here for fileComplete list of GenBank identifiers for all predicted and known toxins and antitoxins. The table contains the GenBank identifiers (GI numbers) for all predicted and known toxins and antitoxins identified in this work in 750 complete prokaryotic genomesClick here for file


We analyzed pertussis reporting, death, hospitalization, and serodiagnostic data from 1976 to 1998 to help explain the cause of the 1996 pertussis outbreak in the Netherlands. The unexpected outbreak was detected by an increase in pertussis reporting and by other surveillance methods. In 1996, according to reporting and serologic data, the increase in pertussis incidence among (mostly unvaccinated) children less than 1 year of age was similar to the increase in hospital admissions. Among older (mostly vaccinated) persons, the increase in hospital admissions was relatively small. The increase in pertussis incidence was higher among vaccinated than among unvaccinated persons of all ages. This resulted in lower estimates of vaccine effectiveness. The proportion of pertussis infections resulting in recognizable symptoms may have increased among vaccinated persons because of a mismatch of the vaccine strain and circulating Bordetella pertussis strains. The small immunogenicity profile of the Dutch vaccine may have resulted in greater vulnerability to antigenic changes in B. pertussis.


Treatment-resistant trigeminal neuralgia is a distressing condition, for both the patient and the treating doctor. To our knowledge, there are no reported cases of trigeminal neuralgia successfully treated with oral sumatriptan in the literature.A 51-year-old Caucasian woman was prescribed opiate analgesia for management of her treatment-resistant trigeminal neuralgia. Given the possible harmful effects of initiating such a course of treatment, a speculative therapeutic trial with oral sumatriptan was initiated with a successful outcome.This case raises the hypothesis that oral sumatriptan may be an effective drug in the treatment of trigeminal neuralgia. Further research is required to test this theory. One of the challenges in primary care is the ongoing management of patients with chronic painful syndromes who have not responded to the standard treatment regimens. Polypharmacy, multiple speciality referrals, patient dissatisfaction, depression, frequent consultations and physician frustration often ensues . Such coThis case highlights a further possible treatment option for trigeminal neuralgia resistant to standard treatment options.We present the case of a 51-year-old Caucasian woman with a four-year history of recurrent, right-sided facial pain, in the distribution of all three branches of the trigeminal nerve. These episodes occurred on average two to three times per week and were characterised by brief episodes of unilateral right-sided sharp lancinating pain, lasting on average less than one minute and predominantly affecting the maxillary and mandibular divisions of the trigeminal nerve, although occasionally involving all three branches. Attacks were typically triggered by such actions as washing her face or smiling. There were no other features associated with these attacks; in particular there were no associated headaches or visual disturbance. She had no prior history of headache and there was no family history of trigeminal neuralgia. Her neurological and dental examinations, a computed axial tomography scan of brain and sinuses, and routine blood tests were all normal.The patient initially commenced on gabapentin (up to 1200 mg daily) to which carbamazepine (up to 800 mg daily) was later added without any significant benefit. Following a neurosurgical assessment, she underwent decompression of her right trigeminal nerve, nine months after the initial onset of her symptoms. Unfortunately, her symptoms worsened following this and she was referred for radiofrequency ablation 14 months after the decompressive surgery. This resulted in some permanent numbness in her right infra-orbital area but no pain relief. She was then referred to a specialist pain clinic and initially treated with a combination of dothiepin (75 mg daily), pregabalin (600 mg daily) and numesulide (200 mg daily). This regime resulted in a transient improvement in her symptoms. At this stage, oxycodone 10 mg daily was added; a treatment escalation that her general practitioner (GP) had reservations about. Based on previous experience using sumatriptan for abdominal pain [Headaches and facial pain are common, and patients with these symptoms frequently present to their GP for advice. Careful history taking and physical examination are the mainstay of accurate diagnosis in the evaluation of these patients. The majority of conditions are self-limiting and managed in general practice without the need for referral or further diagnostic tests. It can, however, be difficult to distinguish between migraine, trigeminal neuralgia, atypical facial pain and cluster headaches with clinical certainty . The symTrigeminal neuralgia is important due to its high morbidity. It usually presents after the age of 50, is unilateral and affects the sensory branches of the trigeminal nerve . This isSumatriptan is a serotonin (5-HT) agonist, specifically developed to relieve migraine headaches. Although the cause of migraine is not fully understood, it is thought that a widening of blood vessels in the brain causes the throbbing pain of migraine headaches. Sumatriptan works by causing vasoconstriction of these vessels via the stimulation of serotonin (or 5-HT) receptors. Naturally occurring serotonin normally acts on these receptors, causing blood vessels in the brain to narrow. Sumatriptan mimics this action of serotonin by directly stimulating the serotonin receptors in the brain. This results in narrowing of the blood vessels and in effective relief of the migraine headache pain. A Cochrane review has confirmed the effectiveness of sumatriptan in the acute management of migraine .Propranolol, a beta-blocker, is recognised as effective medication for the prophylaxis of migraine  and is cThe question of diagnosis in this patient is critical, given the response to treatment. It is tempting to take the view that this patient has an atypical form of migraine, based on her response to both specific anti-migraine medication (sumatriptan) and a proven anti-migraine prophylaxis medication (propranolol), and that the original diagnosis of trigeminal neuralgia was incorrect. However, the clinical presentation of her symptoms strongly supports a diagnosis of trigeminal neuralgia, given the very brief duration of the episodes, the absence of any other symptoms other then unilateral pain in the distribution of the trigeminal nerve and the presence of clear physical triggers, such as washing her face, precipitating an attack. Moreover, a diagnosis of trigeminal neuralgia was felt to be the correct diagnosis by an experienced general practitioner, a neurologist and a neurosurgeon who advocated and carried out invasive decompressive surgery and radio-ablation which would be wholly inappropriate if any doubt had existed concerning the diagnosis. Given this, a more plausible explanation is that the vasoconstrictive mechanism of the sumatriptan relieved whatever compressive or irritative effects were occurring at the root of the affected trigeminal nerve. Such speculations highlight the fundamental problems in drawing conclusions from such 'trials of therapy'. A hypothesis has arisen and needs to be tested using more rigorous and valid research methodology.In conclusion, based on the experience of this case and a review of the current specialist literature, we advise that GPs, when treating patients with difficult to manage trigeminal neuralgia, consider a trial of oral sumatriptan and report their findings.The authors declare that they have no competing interests.Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.JM was the patient's primary care physician. Both JM and AN contributed equally to the design, literature review and drafting of this case report. JM and AN have both seen and approved the final submitted manuscript.


Villoglandular papillary adenocarcinoma (VPA) is a very rare subtype of adenocarcinoma of the uterine cervix, but a well-recognized variant of cervical adenocarcinoma with a favorable prognosis and generally occurring in women of child-bearing age. Only five cases of VPA and pregnancy have been reported. Herein, we report a case of VPA diagnosed during early pregnancy and managed successfully with conservative measures; our patient delivered a healthy baby in full term. A successful pregnancy can be completed in patients with VPA without lymph-vascular invasion, when treated conservatively. This management is particularly desirable in young women to preserve reproductive capability. The incidence of cervical adenocarcinoma is on the rise over the last decades. Villoglandular papillary adenocarcinoma (VPA) is a very rare subtype of adenocarcinoma of the uterine cervix. The true incidence of this form of adenocarcinoma is unknown. The classical histologic appearance of this entity is a surface papillary component of variable thickness with papillae that are usually tall and thin, but occasionally short and broad, with a fibrous stromal core. The tumor cells should have no more than mild-to-moderate nuclear atypicality and scattered mitotic figures. It affects a younger age group and has an excellent prognosis as compared to other endocervical adenocarcinomas , 2. To oA 28-year-old Japanese woman, gravida 1, para 1, was admitted with atypical genital bleeding and underwent polypectomy of the uterine cervix at 9 weeks' gestation. The 1-cm resected polyp was pathologically diagnosed as VPA . The tumWe report an extremely rare case who was diagnosed VPA in the 1st trimester and was managed conservatively throughout the pregnancy with successful results for both the mother and the baby. VPA of the uterine cervix is a rare form of cervical adenocarcinoma first described by Young and Scully in 1989 –11. ThesYoung and Scully recommended careful inspection of the histological specimen and if the villoglandular component is the exclusive or almost exclusive pattern then a diagnosis of VPA can be ascribed . Other pPregnancy associated with VPA of the cervix has been reported in only five cases –7. In twIn conclusion, despite the limited experience of cervical VPA diagnosed during pregnancy, conservative treatment can be successfully achieved in selected patients after a thorough evaluation of the depth of invasion, the lymph vascular involvement, and the association of other carcinoma histologies in conjunction with the VPA (i.e. adenocarcinoma or squamous cell carcinoma).


To the best of our knowledge, this case report describes the longest disease-free interval between primary diagnosis and metastatic recurrence of an osteosarcoma.A 35-year-old Caucasian American man presented with asymptomatic lung metastases 21 years after being diagnosed and treated for lower extremity osteosarcoma. He underwent curative lung resection, but 2 years thereafter developed metastatic disease in the scapula and tibia and, after resection and chemotherapy, is in remission 1 year later.This case highlights the importance of long follow-up periods and continued surveillance of osteosarcoma patients after initial curative treatment. Osteosarcoma is the third most common cancer in adolescence, after lymphomas and brain tumors. One-third of patients who present with osteosarcoma will have recurrent disease. Within the recurrent disease group, 95% of relapses occur within the first 5 years, with the lung as the most common site of metastases. Reported relapses beyond 10 years are rare in the literature. We describe the case of a 35-year-old man who presented with lung metastasis 21 years after undergoing surgical resection and adjuvant chemotherapy for osteosarcoma of the femur. The patient underwent a curative resection. Histological studies described this calcified lesion as osteosarcoma, thereby confirming, to the best of our knowledge, the longest known disease-free interval to metastatic recurrence reported in the English literature. We discuss the relevant clinical knowledge regarding late relapse of osteosarcoma.At the age of 14, the patient was diagnosed with osteosarcoma of the left femur. He received neoadjuvant chemotherapy consisting of doxorubicin and cisplatin, and underwent above-the-knee amputation of his left leg. Adjuvant chemotherapy included doxorubicin, methotrexate and vincristine.Twenty-one years after his initial osteosarcoma resection, the patient presented with cellulitis of his left thigh, which was treated successfully. During his hospital stay, a chest X-ray revealed a mass in the infrahilar area of the patient's right thorax Figure . A compuThe patient underwent curative resection, and pathology of the right middle and lower lung lobes revealed osteosarcoma with extensive chondroblastic differentiation Figure  and 3b. Osteosarcoma is the most common primary malignancy of the bone, with a peak incidence in the second decade and an additional peak of incidence in elderly individuals . Historiet al., which describes local recurrence 20 years after surgery [Late recurrence is usually described as occurring more than 5 years after primary osteosarcoma treatment. However, the recorded cases with the longest time spans before recurrence are all of local recurrence, such as the report by Lau  surgery . The Riz surgery .In 2004, Strauss and colleagues published an article describing eight patients seen at the London Bone and Soft tissue Tumor Service who had relapsed after long disease-free intervals . The artThe most recent study on late recurrence comes from the Rizzoli Institute in 2006 and included 648 patients with non-metastatic osteosarcoma to determine predictive factors for late recurrence [Our patient was initially treated for osteosarcoma with surgical resection and chemotherapy, and presented 21 years later with a single metastasis to the right lung. Osteosarcomas are the least likely bone tumors to relapse late, making this case even more unusual. This case represents the longest disease-free interval to metastatic recurrence in the English literature, and underscores the need for continued surveillance of osteosarcoma patients.The authors declare that they have no competing interests.SG performed the histologic examination of the pathology specimens and was a major contributor in writing the manuscript. SB, SM and AH contributed significantly to the concept, design and writing of the manuscript. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Aspergillus species among immunocompromised patients. Although this infection frequently involves the lung parenchyma, it is unusual to find it limited to the tracheobronchial tree, a condition known as invasive aspergillus tracheobronchitis.Invasive pulmonary aspergillosis is the most common form of infection by Aspergillus species.A 65 year-old Hispanic man from Bolivia with a history of chronic lymphocytic leukemia developed cough and malaise eight months after having an allogenic stem cell transplant. A computed tomography of the chest revealed an area of diffuse soft tissue thickening around the left main stem bronchus, which was intensely fluorodeoxyglucose-avid on positron emission tomography scanning. An initial bronchoscopic exam revealed circumferential narrowing of the entire left main stem bronchus with necrotic and friable material on the medial wall. Neither aspirates from this necrotic area nor bronchial washing were diagnostic. A second bronchoscopy with endobronchial ultrasound evidenced a soft tissue thickening on the medial aspect of the left main stem bronchus underlying the area of necrosis visible endoluminally. Endobronchial ultrasound-guided transbronchial needle aspiration performed in this area revealed multiple fungal elements suggestive of We describe the first case of invasive aspergillus tracheobronchitis in which the diagnosis was facilitated by the use of endobronchial ultrasound guided trans-bronchial needle aspiration. To the best of our knowledge, we are also presenting the first positron emission tomography scan images of this condition in the literature. We cautiously suggest that endobronchial ultrasound imaging may be a useful tool to evaluate the degree of invasion and the involvement of vascular structures in these patients prior to bronchoscopic manipulation of the affected areas in an effort to avoid potentially fatal hemorrhage. Invasive aspergillosis is one of the most common fungal infections in immunocompromised hosts, involving the respiratory tract in 90% of cases . This diA 65-year-old Hispanic man from Bolivia with chronic lymphocytic leukemia (CLL) diagnosed 11 years before presentation and initially treated with alkylating agents, steroids and purine analogs, underwent an allogenic stem cell transplant from an HLA-compatible sibling a year before presentation. He developed cough and malaise eight months after the transplant for which he was admitted to another facility. Chest radiographs were reportedly negative and the patient was diagnosed with bronchitis and sent home on oral antibiotics. He continued to have intermittent cough productive of clear sputum associated with worsening malaise and weakness for about four months and was eventually evaluated at our hospital. Computed tomography (CT) of the chest revealed diffuse soft tissue thickening around the left main stem (LMS) bronchus Figure . The rad3), but not neutropenic .His medical history was remarkable for adult-onset asthma, two episodes of pneumonia several years ago, actinic keratosis and squamous cell carcinoma of the skin. He was a lifelong non-smoker and was on immunosuppression with prednisone and tacrolimus, and routine prophylaxis with atovaquone, valacyclovir and voriconazole. His vital signs were stable and his respiratory exam was unremarkable. He was thrombocytopenic  ,8,9. Theet al.  reportedet al. ,8.The clinical mainfestations of IATB are entirely different from those of invasive pulmonary aspergillosis. The insidious presentation with non-specific symptoms and the paucity of findings in chest roentgenograms often delay the diagnosis, giving this disease an ominous prognosis -12. AirwAspergillus hyphae in special stains or by recovering the organism in fungal cultures. The samples obtained during the first bronchoscopic examination of our patient, however, were negative. As the patient's thrombocytopenia was refractory to platelet transfusions, we opted for EBUS-guided TBNA in order to achieve diagnosis with the lowest risk of bleeding. This is the first case of IATB in which diagnosis was facilitated by EBUS that we could find in the medical literature. It is our opinion that real-time EBUS might also be useful in delineating the relationships during fungal invasion into adjacent tissues and the involvement of major vascular structures, potentially preventing lethal hemorrhage. This type of infection can progress very rapidly, leading to invasion of major vessels in the time between CT imaging and bronchoscopy, making routine bronchoscopic manipulation and sampling of debris extremely dangerous and even fatal [The diagnosis of IATB is almost always confirmed by bronchoscopic examination and sampling. Although thrombocytopenia is commonly a limiting factor for acquiring endobronchial biopsies, bronchoscopic aspiration of debris and bronchial washings allow diagnosis in the majority of cases by showing the presence of en fatal .In conclusion, IATB is a rare form of invasive aspergillosis affecting mainly immunocompromised patients. The non-specific clinical presentation often leads to late diagnosis and poor prognosis. We report the first case of IATB diagnosed by EBUS-guided TBNA. We also cautiously suggest that EBUS imaging may be a useful tool to evaluate the depth of fungal invasion into adjacent tissues and the involvement of vascular structures in these patients prior to bronchoscopic manipulation of the affected areas in an effort to avoid fatal hemorrhage.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the journal's Editor-in-Chief.The authors declare that they have no competing interests.RA performed initial bronchoscopic exam. RFC, CAJ, MS, RM and GAE performed EBUS. All authors equally contributed to the creation of the manuscript.