The authors' experience with experimentally produced femoral capital osteonecrosis in rats is reviewed: incising the periosteum at the base of the neck of the femur and cutting the ligamentum teres leads to coagulation necrosis of the epiphysis. The necrotic debris is substituted by fibrous tissue concomitantly with resorption of the dead soft and hard tissues by macrophages and osteoclasts, respectively. Progressively, the formerly necrotic epiphysis is repopulated by hematopoietic-fatty tissue, and replaced by architecturally abnormal and biomechanically weak bone. The femoral heads lose their smooth-surfaced hemispherical shape in the wake of the load transfer through the hip joint such that, together with regressive changes of the joint cartilage and inflammatory-hyperplastic changes of the articular membrane, an osteoarthritis-like disorder ensues.Diverse therapeutic options are studied to satisfy the different opinions concerning the significance of diverse etiological and pathogenic mechanisms: 1. Exposure to hyperbaric oxygen. 2. Exposure to hyperbaric oxygen and non-weight bearing on the operated hip. 3. Medication with enoxaparin. 4. Reduction of intraosseous hypertension, putting to use a procedure aimed at core decompression, namely drilling a channel through the femoral head. 5. Medication with vascular endothelial growth factor with a view to accelerating revascularization. 6. Medication with zoledronic acid to decrease osteoclastic productivity such that the remodeling of the femoral head is slowed.Glucocorticoid-related osteonecrosis appears to be apoptosis-related, thus differing from the vessel-deprivation-induced tissue coagulation found in idiopathic osteonecrosis. The quantities of TNF-α, RANK-ligand and osteoprotegerin are raised in glucocorticoid-treated osteoblasts so that the differentiation of osteoclasts is blocked. Moreover, the osteoblasts and osteocytes of the femoral cortex mostly undergo apoptosis after a lengthy period of glucocorticoid medication. Osteonecrosis of the femoral head is of both clinical and economic interest, nearly 20,000 patients being hospitalized annually in the U.S.A. for treatment of this disease. Different risk factors have been discussed, yet the etiology and the pathogenesis of osteonecrosis are still uncertain . ClinicaRodents are frequently used in preclinical tests of novel therapeutic modalities. So it behooves the reader to notice that interrupting the circulation in the femoral head of rats, with their lifelong persisting physeal cartilage, mimics children's Legg-Calvé-Perthes disease more than it resembles adult osteonecrosis . IrrespeThe effects of therapeutic interventions on the course of osteonecrosis of the femoral head may be studied using various models. The following model has been applied by the authors of this review: the blood supply and drainage of epiphysis are interrupted by cutting the ligamentum teres and incising the periosteum at the cervical base of the femoral head of 6 month-old rats. After the operation, the rats are placed in spacious cages such that their perambulation is almost unhindered. At the time of sacrifice, the femora are excised and fixed in formalin. The samples are embedded in paraffin after decalcification. Blocks are cut such that longitudinally orientated sections bisect the insertion of the ligamentum teres [Necrosis of the adipose and hematopoietic cells is histologically evident as early as the 2nd postoperative day. Necrosis of the subchondral and trabecular bone first becomes overt on the 5th postoperative day. Repair begins soon afterwards with growth of viable tissue from the epiphyseal-capsular junction into the necrotic debris within the intertrabecular spaces. Residues of the eosinophilic, granular, necrotic marrow are no longer apparent after the 3rd week. Undifferentiated mesenchymal cells initially infiltrate the necrotic marrow and are later replaced by well-vascularized fibrous tissue, carrying with it macrophages, resorbing the dead soft tissues, and by osteoclasts, absorbing the necrotic bone. Beginning in the 3rd postoperative week, newly-formed intramembranous and appositional bone remodel the original osseous framework of the epiphysis. Unevenly contoured, recently formed bony beams crisscross the intertrabecular fibrous tissue, spanning between the viable osteoid seams and the dead trabeculae. Complete replacement of all the necrotic by living bone occurs at the 6-week interval or later. The marrow spaces are repopulated by hematopoietic-fatty tissue. The femoral heads collapse, flatten or are otherwise disfigured. The physeal cartilage is mostly unaffected. Fibrous tissue invades the joint cartilage wherever the continuity of the subchondral bone plate is disrupted. Chondroclasts erode the cartilaginous matrix. A fibrous pannus eventually covers the roughened and fibrillated surface cartilage. As judged by the lack of stainable chondrocytic nuclei, the articular cartilage is undergoing focal chondrolysis, resulting occasionally in delamination of a partly free-floating cartilaginous membrane. The tissue in the expanded joint capsule is contiguous with the pannus and fibrous tissue in the marrow spaces. A shortcoming of this model is the widespread necrosis of the rat femoral heads, sporadically extending to the articular and physeal cartilage [Why is the epiphysis of the femoral head frequently affected by ischemic insults, while the diaphysis and metaphysis are spared? According to Johnson and her colleagues, the limited blood circulation accounts for the clinically high incidence of osteonecrosis of the femoral head . Blood sThe gradual substitution of necrotic by living bone is divided into phases, which nevertheless overlap. Oxygen- and nutrient-deprived osteocytes and marrow cells die to the nearest link with the collateral circulation. Neutrophilic infiltration characterizes the acute phase, which is rapidly followed by the chronic stage during which invasion of macrophages is dominant. Granulation tissue forms and, with time, the detritus is resorbed. The stage of repair starts with the lessening of inflammation and resorption of the dead tissues. It is set in motion by an influx of pluripotential mesenchymal cells. The environmental variations and stresses to which the cells are exposed induce the pluripotential cells to differentiate into fibroblasts, chondroblasts, osteoblasts or angioblasts. The bulk of the cells involved in the reparative process infiltrate the necrotic femoral head from the hyperplastic subsynovial layer. Repair is associated with an ingress of capillary buds, which are recruited by vascular endothelial growth factor (VEGF) and diverse cytokines, which are abundantly synthesized by and released from the synovial fibroblasts residing within the hyperplastic subsynovial cell population ,11.The cues that monitor the behavior of the mesenchymal cells are probably derived from the microenvironment. To exemplify, cartilage and bone are produced in areas of low and high oxygen tension, respectively. Afterwards, the cartilage is transformed by endochondral ossification into bone . EventuaThe above-described repair of the necrotic epiphyses might suggest that the healing process restores the femoral heads to their former selves. Yet unless the necrotic segment is small or restricted to the non-load bearing part of the femoral head, this is not the case in man. The clinical condition of untreated patients goes gradually downhill. Inasmuch as the reparative properties of healthy bone are excellent, this apparent discrepancy remains to be elucidated.Sevitt pioneered the prevailing explanation of avascular necrosis of the femoral head in the wake of a fractured femoral neck and the ensuing revascularization of the epiphysis . In the With the ingrowth of blood vessels, the reparative processes are launched by permeation of circulation-born monocytes throughout the necrotic debris. The emigrating monocytes proliferate and, having differentiated into macrophages and osteoclasts, resorb the dead tissues. Perivascular progenitor cells transform into osteoblasts, which deposit bone. In addition, the invading tissues are replete with undifferentiated mesenchymal cells, which stay dormant awaiting appropriate signals, upon which they are induced to proliferate and differentiate into fibroblasts, chondroblasts, osteoblasts, angioblasts and lipoblasts. This cascade of events, however, does not hold true for all species; thus, the undifferentiated cells in canine experimental osteonecrosis migrate first and foremost from adjacent living bone .As stated above, recently deposited and mineralized bony matrix is biomechanically inferior to mature bone in respect of stiffness, strength, and toughness. Hence, the recently remodeled femoral heads do not withstand the transarticular stresses of daily activity-related loads without caving -20. As rThe restorative activities begin during the 2nd postoperative week in the rat model. The near-hemispherical, smooth-contoured profile of the healthy femoral head is lost by the 2nd to the 3rd postoperative month. The femoral heads deviate structurally from normal shape in that they acquire a crescent-profiled, triangular or other aberrant form with rugged, murky brown joint cartilage. Not infrequently, there are no residues of dead tissues at this point in time. The amounts of newly deposited bone vary from one segment of the epiphysis to another and from one rat to another. Variously shaped and sized, lamellar-fibred or woven-fibred, newly formed trabeculae of bone crisscross the loosely to densely textured fibrous tissue that has permeated the intertrabecular spaces Fig. . CuboidaThe articular aspect demonstrates a spectrum of changes ranging from a reduced content of glycosaminoglycan in the cartilage to a segmentally burnished and eburnated bony surface devoid of cartilage Figs.  and 2. TDead bone retains its rigid qualities for quite a long time unless it is substituted by newly formed osseous tissues. Non-remodeled necrotic bone should theoretically retain its properties of resistance to load-bearing and bending strains. There is consequently no biomechanical basis for evolving alterations of the conformation of the necrotic femoral head in the immediate period after an ischemic injury. In fact, descriptions in the literature of both functional and morphological deviations from the norm of the postosteonecrotic rat femoral head pertain to the late stage of the disease.Human analyzers adequately and competently interpret what they perceive, but they experience difficulties in quantifying what they observe . This knThe distortion of an infarcted femoral head depends on the extent of structural degradation of its cancellous bone . BecauseThe maximal deficit in material properties manifests itself during the mid- to late-stages of the repair phase , which iInterestingly, about one third of the femoral heads of children with Perthes disease round up . The epiContrary to the universally accepted paradigm, the mode by which the rats' vessel-deprived necrotic femoral heads remodel is unanticipated. The height-to-width ratios of numerous epiphyses obtained 18 and 36 days postoperatively are in fact greater than those of the control epiphyses. The adaptive reshaping of osseous tissues is responsive to alterations in the distribution and magnitude of the strain generated within the bone . ComprisCuretting the core of the necrotic epiphysis (thus stimulating osteoneogenesis) is assumed to prevent the collapse of the joint surface following blending with the subchondral bone plate of a cancellous bone-augmented vascularized fibular graft . LikewisIt is currently conjectured that, firstly, vascular impediment and defective repair capacity act in concert in causing non-traumatic variants of osteonecrosis and, secondly, the replicative potential of the osteoblasts is reduced in the living parts of the femoral head, supporting the pathogenetic role in osteonecrosis of malfunctioning of the bone-forming cells ,39.Taking for granted the accuracy of the paradigm of the pathogenetic role of vascular deprivation and anoxia in bringing about necrosis of the femoral head, revascularization and oxygenation ought to be the paramount therapeutic modalities. As a matter of fact, both core decompression and implantation of a vascularized bone graft have met with success in rescuing patients' necrotic femoral heads. This success is attributed, at least partly, to the encouragement of ingrowth of well-vascularized fibrous tissue into the necrotic bone. The size of the necrotic zone dictates the fate of necrotic femoral heads. In rats, resorption of the epiphysis takes place at all times because their femoral heads undergo total necrosis because the blood inflow and outflow at the cervical level and ligamentum teres are completely severed ,41. CoreIn the experience of Simank et al., drilling a sheep's epiphysis (their model of core decompression) encouraged healing of the necrotic femoral heads . The autLancing the epiphysis with a 21-gauge needle is not expected to weaken the bone. An explanation for the conduit-related intensification of remodeling should, therefore, be sought elsewhere. 1. Conceding that a conduit accelerates the healing process as a result of its tension-lowering effect and opening up a path for vascular ingrowth, the rapid replacement of dead by living bone leads to the deposition of a weak osseous structure that is unlikely to carry weight-bearing loads without collapsing. 2. The conduit hastens the development of osteoarthritis since the osteochondral junction is inadequately reconstructed. 3. The insertion of a needle through the foveola into the epiphysis creates an inlet that permits the synovial fluid to spill from the joint cavity into the intertrabecular spaces, thus delaying the repair of bone defects. 4. The synovial fibroblasts in the distended joint capsule of rats with vessel-deprived osteonecrosis of the femoral head are jam-packed with vascular endothelial growth factor. The overexpression of this and other intermediates probably accounts for the enhanced ingrowth of blood vessels after the creation of an intraosseous conduit in the necrotic femoral heads ,45,56.The expectation that anticoagulation would thwart osteonecrosis of the femoral head goes back to the early 1970s, when Fahlström et al. established that the incidence of osteonecrosis complicating fractures of the femoral neck was reduced nearly fourfold in patients on a daily heparin regimen as compared to a control group of untreated patients . Study oA series of hyperbaric oxygen-treated patients with osteonecrosis of the femoral head was reported in 1990 at the 10th International Congress of Hyperbaric Medicine . However2 is employed. The in vitro upregulation of osteoclastic activity may be related to the extended exposure to O2 radicals. In vivo, sustained hyperoxygenation results in the production of a repair tissue replete with structurally weak collagen fibers [Daily exposure of patients with Steinberg stage-I osteonecrosis to HBO for 100 days reportedly results in the return to a normal MRI pattern in ~80% of cases. This cure rate compares favorably with a ~80% rate of collapse of the femoral heads in untreated patients within 4 years of the onset of the disease . Yet then fibers -62. Too n fibers . The bren fibers . Ingrowtn fibers . To sum Daily 90 minute exposures to HBO in a monoplace hyperbaric chamber enhances osteogenesis in rats after ischemic damage of the femoral heads. Hyperoxygenation is intended to uphold the innate re-establishment of well-being, and to enhance fibrogenesis, appositional and intramembranous osteogenesis, resorption of necrotic soft tissues and osteoclastic osteolysis during the late phase of osteonecrosis ,67.Histomorphometric parameters indicate that exposure to HBO modifies the architectural distortion of the femoral heads . The HBOExposure to HBO provides an optimal environment for repair processes as the additional oxygen carried by the circulation to ischemic sites raises the oxygen tension in the tissues. The hyperoxygenation-mediated relief of ischemia boosts the activities of fibroblasts, osteoblasts and osteoclasts in addition to supplying the extra oxygen that is indispensable for meeting the increased metabolic demands of regenerating tissues. Given that vascularization of the ischemic site is sufficient, exposure to HBO within the first 4 to 6 hours after injury achieves the optimum results -70. ShifThe prognosis after conservative therapy of femoral capital osteonecrosis is mostly poor, osteoarthritis more often than not evolving within 2 to 3 years of the diagnosis . A perfeIt is now close to half a century since HBO was first acclaimed as a beneficial adjunct to conventional therapy for miscellaneous illnesses -80. An iIn a study of the combined effect of exposure to HBO and NWB on the repair of necrotic femoral heads, rats were housed in an enclosed 2 × 2 × 1.3 feet Plexiglas space, in which the hind limbs were suspended by tail traction so that the hip joints were not loaded. The trailing end of a Velcro strip, wrapped around the rats' tails, was fixated to a crossbar with a wheel and swivel assembly riding on opposite walls of the cage. Thus, the rats had freedom of movement in the longitudinal and orthogonal directions and access to food and water at all times. From the 5th postoperative day, the rats were exposed to 100% oxygen at 2.5 ATA over 22 or 32 sessions, each lasting 90 min. Control animals were treated only by NWB. The rats were killed 30 or 42 days postoperatively. There were no changes in the femoral heads of sham operated (control) rats that had been subjected to NWB, HBO, or both. The gamut of post-osteonecrotic repair activities was enhanced in rats on the HBO plus NWB regimen: osteogenesis, florid osteoblastic rimming, preosteoblasts abutting on necrotic or lately deposited bone, clustered undifferentiated mesenchymal cells in hypercellular fibrous tissue, osteoclastic osteolysis of viable and necrotic bone, chondroclastic chondrolysis and degeneration of the joint cartilage were significantly more advanced than in other reported models of therapy. Severe distortion of the femoral heads ensued in almost a third of the rats. The structural deformations manifested various configurations affecting the shape, symmetry, organization of the hard and soft tissues, and the height as well as the width of the epiphysis. The irregularly shaped femoral heads had jagged surfaces subsequent to asymmetrical resorption of the necrotic bone and erratic substitution by thriving, recently formed bone. In place of the innate, smoothly surfaced hemispherical outline of the femoral head, any of a myriad geometric configurations evolved. Loss of tissue led to localized surface depressions, which were lined by a layer of synovial-like cells several cells thick. In other instances, exuberant tissue proliferation resulted in an elevation of the articular aspect. The sporadically decreased epiphyseal height signified flattening of the bony compartments of the femoral heads. Even though remodeling and distortion often coincided, the hemispherical profile of the femoral head was every so often preserved. Where sizable parts of the epiphysis had been replaced, the cartilage, the bone, the fibrous tissue, or all of these were always accompanied by peculiar architectural modifications. The semiquantitatively gauged parameters indicating deformation were statistically less significant on the 30th postoperative day in rats treated by the combined NWB plus HBO regimen than in the rats treated with either NWB or HBO alone ,44,59,77Several studies have established the favorable effects of HBO therapy on the course of certain ischemia-induced conditions, but there is no consensus about its therapeutic value in osteonecrosis of the femoral head .2 which, after HBO medication, increases the diffusion distance notwithstanding the fibrous tissue enclosure. The hyperoxygenation-induced relief of marrow edema is a spin-off of HBO exposure; it is the byproduct of reflex vasoconstriction and oxygen-induced osmosis, which reverses the usual pumping mode of interstitial fluids, i.e. from the tissues back into the circulation. Hyperoxygenation also induces the precursors of the multipotential mesenchymal cells to mature into osteoblasts and at the same time encourages osteoclastic osteolysis such that remodeling is enhanced overall [Vessel-deprived epiphyseal osteonecrosis in rats does not fully imitate all the clinical, humoral and metabolic conditions that precede the disease in man. Nevertheless, the causal pathway of impeded blood supply and drainage is embodied in most experimental models of the disorder ,89. The  overall -96. Fina overall . Acting  overall .VEGF stimulates angiogenesis, recruitment and migration of osteoblasts and activation of osteoclasts. So far so good; but medication with VEGF would also enhance the removal of dead bone and increase the formation of a mechanically weak intramembranous bone, two events that ought to be avoided at all costs. In the context of fracture healing, a slow VEGF-releasing device is an effective therapeutic mode -102, butLieberman et al. recommended combining core decompression with VEGF medication so as to strengthen "patients' angiogenic potential" . This prLittle et al. have carried out a proficient series of experiments on the medication of rats with zoledronic acid (ZA) after surgically inducing osteonecrosis of the femoral head. They hypothesize that this bisphosphonate may preserve the structure of the femoral head while, at the same time, allowing incremental bone repair. Indeed, treatment and prophylaxis with ZA improve the sphericity and maintain the architecture of the necrotic femoral head. They have studied rats medicated subcutaneously with saline, ZA at one and 4 weeks after the operation (ZA-post), and ZA at 2 weeks pre-operation and at 1 and 4 weeks post-operation (ZA-pre-post). Six weeks postoperatively, 71% of the femoral heads of the saline-treated rats were aspherical. This contrasts with 13% and 0% aspherical femoral heads 6 weeks postoperatively in the ZA-post and ZA-pre-post animals (p < 0.05). Histomorphometrically, the bone volume was decreased by 12% in the saline group and close to 20% in the ZA-post and ZA-pre-post groups (p < 0.05). The retention of necrotic bone in the epiphyses of the treated rats was unambiguous. The difference between the non-treated and treated rats is explicitly due to the reduction in bone turnover .Hip osteoarthritis is the leading treatment failure in children with Perthes disease and in adults with osteonecrosis. It results from the abnormal load transfer from the acetabulum to the femur across a remodeled and deformed femoral head. Contrary to clinicians' precepts, therapy that minimizes or hinders the remodeling processes delays the progressive deterioration of the articular structures. A balance between osteolysis and osteogenesis in the appropriate ratio is decisive in forestalling the collapse of the epiphysis, as preservation of the hemispherical shape of the femoral head is crucial in averting the development of osteoarthritis ,62.The means of treating osteonecrosis of the femoral head appraised in this review have been under experimental and clinical analyses for a few decades. Each of them has been praised at one time or another for providing the solution to the orthopaedic surgeons' frustrating deadlock in respect of restoring the necrotic femoral head to its earlier physical condition. In the rat model of vessel deprivation-induced osteonecrosis of the femoral head, medication with enoxaparin, construction of an intraosseous conduit, exposure to HBO and exposure to HBO plus NWB have been shown to hasten those reparative activities that are conventionally accepted as the epitome of revitalization of avascular dead bone. Investigators have a priori endeavored to enable vascular ingrowth. Accepting that osteonecrosis is caused by lack of blood supply, it is reasoned that the sooner the vasculature is reinstituted and delivery of oxygen and nutrients is returned to normal, the faster and more comprehensive would be the reconstruction of a living and mechanically well-performing femoral head. In mild cases, the femoral heads more or less retain their hemispherical profile. In more advanced cases, they are somewhat flattened or otherwise deviate from the hemispherical shape. Lastly, in the most severe cases, the femoral heads acquire any of a number of bizarre geometric forms. All repair processes are accelerated in rats treated by the above-mentioned means, including amassing undifferentiated mesenchymal cells, and profuse fibrogenesis, vasculogenesis, chondrogenesis and osteogenesis. At first sight it appears that all the clinically desired goals are attained. Alas, the profile of many a treated rat femoral head is disfigured to such a degree that smooth functioning of the hip joint is out of the question. The rising array of deformations correlates with an increasing extent of repair, indicating an inverse relationship between the degree of reconstruction and extent of revascularization on the one hand, and the magnitude of distortion of the femoral heads on the other. This explains the dictum that rats with maximally reconditioned necrotic femoral heads have the worst of it .Brown et al. have given an account of the biomechanical properties of cancellous bone samples obtained from middle-stage and late-stage osteonecrosis of adult necrotic femoral heads. Compared to specimens retrieved from femoral heads of healthy individuals, samples removed from infarcted zones exhibit low yield strength, a much reduced elastic modulus and a modestly increased strain-to-failure. It is noteworthy that minor deviations in the strength and stiffness of bone taken from the affected regions are associated with large differences in the pattern of collapse and revascularization of the femoral heads . An orthThe clinical relevance of animal experiments utilizing hyperoxygenation as the exclusive mode of treatment may be criticized because HBO in the clinical setting constitutes an adjunct to other therapeutic modalities. Incidentally, the outcome of studies of exposure of spontaneously hypertensive rats to HBO is irrelevant to our subject matter, because hyperoxygenation is utilized prophylactically . As a ru2, in so far as too much osteolysis in too short a time may result in an untimely collapse of the femoral head [Rats with vessel-deprived osteonecrosis of the femoral heads do not gain markedly from a NWB regimen. This concurs with the almost predictable collapse of the necrotic femoral heads in patients managed by restricted weight bearing . While Nral head ,108,109.ral head ,111.The experimental mimickers of osteonecrosis of patient femoral heads possess certain distinctive traits, which differ from the disease as witnessed at the bedside. Osteonecrosis, with only a few exceptions, affects only part of the femoral head, while the epiphysis of rats virtually always undergoes total necrosis. Also, glucocorticoid-induced osteonecrosis in patients does not duplicate the coagulation-type death of the vascular deprivation-induced disorder in rats, but rather evinces an apoptotic process . HoweverIt is well known that glucocorticoids suppress osteogenesis and cause calcium loss. Yet the mechanism(s) linking glucocorticoid medication to osteonecrosis of the femoral head remain mystifying. It is fascinating that idiopathic and glucocorticoid-induced osteonecrosis cannot be distinguished histologically, maybe because at least some intermediate events are common to both . Fat embThe ratio of cytokines promoting the differentiation of osteoclasts to those blocking this process is raised in glucocorticoid-treated osteoblasts. These cytokines include the TNF-α family, RANKL and osteoprotegerin ,118. It It is challenging to determine how glucocorticoids bring about widespread osteolysis on the one hand, and cause damage to distinctive skeletal sites – say, the femoral head – on the other. Experimental data imply that glucocorticoid-induced apoptosis of osteocytes coincides with vascular blockage-independent osteonecrosis . By itseGlucocorticoid-promoted expression of the RANK-ligand by the osteoblasts represents a central pathway in osteoclast maturation . The actGlucocorticoids play a prominent physiological role in the turnover of healthy bone. The process of osteoblastic maturation in tissue cultures is stimulated by glucocorticoids at the proper concentration ,126. In 2, generated by mechanically strained osteoblasts and osteocytes, are likely to affect the response to glucocorticoids [Trabecular bone bends under a load to a greater extent than cortical bone. Hence, the especial sensitivity of trabecular osteoblasts and osteocytes possibly reflects variations in the microenvironment. Lastly, proapoptotic signals such as NOrticoids . Based orticoids .


The advent of live-attenuated vaccines against measles virus during the 1960'ies changed the circulation dynamics of the virus. Earlier the virus was indigenous to countries worldwide, but now it is mediated by a limited number of evolutionary lineages causing sporadic outbreaks/epidemics of measles or circulating in geographically restricted endemic areas of Africa, Asia and Europe. We expect that the evolutionary dynamics of measles virus has changed from a situation where a variety of genomic variants co-circulates in an epidemic with relatively high probabilities of co-infection of the individual to a situation where a co-infection with strains from evolutionary different lineages is unlikely.We performed an analysis of the partial sequences of the hemagglutinin gene of 18 measles virus strains collected in Denmark between 1965 and 1983 where vaccination was first initiated in 1987. The results were compared with those obtained with strains collected from other parts of the world after the initiation of vaccination in the given place. Intergenomic recombination among pre-/early-vaccination strains is suggested by 1) estimations of linkage disequilibrium between informative sites, 2) the decay of linkage disequilibrium with distance between informative sites and 3) a comparison of the expected number of homoplasies to the number of apparent homoplasies in the most parsimonious tree. No significant evidence of recombination could be demonstrated among strains circulating at present.We provide evidence that recombination can occur in measles virus and that it has had a detectable impact on sequence evolution of pre-vaccination samples. We were not able to detect recombination from present-day sequence surveys. We believe that the decreased rate of visible recombination may be explained by changed dynamics, since divergent strains do not meet very often in current epidemics that are often spawned by a single sequence type. Signs of pre-vaccination recombination events in the present-day sequences are not strong enough to be detectable. Paramyxoviridae family in the order of Mononegavirales and only a single serotype is known. It is among the most infectious viruses known for humans, and no other host species has been identified. Only human populations of a considerable size are able to sustain circulation . The. TheMonoCo-infection of host cells with phylogenetically distinct virus strains is required for recombination events to be detectable in sequence surveys. As a result of the global vaccination against measles a situation of endemic co-circulation of multiple strains ,6,15 shipre-/early-vaccination era isolates used for isolates collected in Denmark during the period of 1965–83 is meant to reflect that these isolates are from a period when vaccination against measles was not practiced in Denmark but was gradually becoming common practice in many other countries in the World. Thus, it cannot be excluded that vaccination in other countries influenced measles virus strains circulating in Denmark at the time, but it is anticipated that these isolates still bear valuable information on the nature of strains circulating before an influence of vaccination was imposed.The term Table Figure Figure Figure One way to distinguish whether incompatibilities are caused by parallel mutations (i.e. true homoplasies) or by recombination is to correlate linkage disequilibrium (LD) between sets of informative sites to distance. Recombination causes a decrease in linkage disequilibrium and more recombination is expected between sites that are further apart from each other. The block structure of close sites is visualised in a different way in Figure effective sites number . The. The35]]2 test. An estimated rate of ρ = 15.8 corresponds to that an expected 45 recombination events have happened in the ancestral history of the 18 pre-/early-vaccination sequences. The estimated recombination rate appears smaller than rates reported for HIV and other viruses [Given these different lines of evidence for recombination, it is of interest to try to estimate the rate of recombination needed to explain the data. The most appropriate method is the finite site, composite likelihood approach implemented in LDhat . The res viruses . The est viruses .In conclusion, the analysis of pre-/early-vaccination era MV sequences shows evidence of recombination at rates important to the evolution of MV. The five different tests of recombination should not be considered independent tests and some of the results might be explained by alternative mechanisms such as convergent evolution of functionally important sites and rate heterogeneity of synonymous variation. However, all tests agree and provide evidence of recombination both through an excess of apparent homoplasies compared to the expected frequency of parallel mutations, and through a decrease in LD with distance, which is difficult to explain by any other hypothesis than recombination. Furthermore, it is difficult to imagine a mechanism other than recombination by which apparent homoplasies could occur pairwise or in triplets in distant parts of the sequence considering also that such patterns are not seen in the post-vaccination data set.The evidence of recombination among pre-/early-vaccination era MV strains and the lack of detection of recombination among post-vaccination era MV strains are consistent with the shift in epidemiology from a situation of co-circulation of strains in populations to a bottleneck situation with incidental introduction of a single strain to a susceptible sub-population of a geographical region. Given that intergenomic recombination and co-infection of individuals are common phenomena of MV it might be assumed that the lineages surviving till today did emerge from a pool of recombining pre-vaccination era strains. A lower level or absence of recombination due to changed epidemiology since then has erased our ability to detect recombination in a global sample of present day MV despite its high level of variability. It is possible that the Danish pre-/early-vaccination era strains are representatives of the very pool in which recombination took place while present-day MV strains are representatives of temporally and/or geographically separated lineages. Analysing informative sites in other parts of the MV genome of present-day lineages render it unlikely that these lineages could have derived from a clonal population structure of a global pool of MV strains .Paramyxovirinae is a nucleocapsid complex in which each nucleocapsid monomer (N) is predicted to be associated by hydrophobic bonds with 6 nucleotides in such a way as to resist non-ionic detergent and high salt and to protect the RNA from RNase digestion [The template for replication in members of the igestion . This tiMeasles virus appear to possess the ability to recombine but the present-day epidemiology of the virus where different sequence types rarely or never meet make the impact of recombination on the distribution of sequence diversity negligible. However, in the prevaccination area, endemic MV allowed more divergent strains to meet and recombine. The present-day strains are thus descendants of recombined sequences but the signal of the early recombination is lost in present-day sequences.AJ417850-AJ417867) [AF410986) , MVi/Montreal.CAN/19.98  , MVi/Vic.AU/12.99  [AF243851) [AY184218) , and China94-1  [The Danish pre-/early-vaccination sequences consist of an 800 base-pair region (nt. 659 to 1458) of the hemagglutinin coding region of 18 strains collected in Denmark, Greenland and the Faeroe Islands between 1965 and 1983 (GenBank accession numbers J417867) . Post-vaJ417867) . The remJ417867) , MVi/AlbY127853) , (Type GF243851) , MVi/GreF045203) .The alignment of the 18 pre-/early-vaccination and 40 post-vaccination sequences does not contain any gaps. The computer program DnaSP 3.99  was usedR2 or the standardized measure D') was investigated following [Recombination was examined using five different, but complementary, approaches. (i) A graphical method by which sets of sites in strong linkage disequilibrium (LD) were visually identified and marked with different colourings Fig. . It was ollowing  and estiollowing . Analysiollowing , using tollowing . This prollowing , using Pollowing .CHM, CPM and LSC collected the data. LSC formulated the hypothesis, and MHS performed all analyses. MHS and LSC wrote the paper.


Many types of research on dementia and cognitive impairment require large sample sizes. Detailed in-person assessment using batteries of neuropyschologic testing is expensive. This study evaluates whether a brief telephone cognitive assessment strategy can reliably classify cognitive status when compared to an in-person "gold-standard" clinical assessment.The gold standard assessment of cognitive status was conducted at the University of Southern California Alzheimer Disease Research Center (USC ADRC). It involved an examination of patients with a memory complaint by a neurologist or psychiatrist specializing in cognitive disorders and administration of a battery of neuropsychologic tests. The method being evaluated was a multi-staged assessment using the Telephone Interview of Cognitive Status-modified (TICSm) with patients and the Telephone Dementia Questionnaire (TDQ) with a proxy. Elderly male and female patients who had received the gold standard in-person assessment were asked to also undergo the telephone assessment. The unweighted kappa statistic was calculated to compare the gold standard and the multistage telephone assessment methods. Sensitivity for classification with dementia and specificity for classification as normal were also calculated.Of 50 patients who underwent the gold standard assessment and were referred for telephone assessment, 38 (76%) completed the TICS. The mean age was 78.1 years and 26 (68%) were female. When comparing the gold standard assessment and the telephone method for classifying subjects as having dementia or no dementia, the sensitivity of the telephone method was 0.83 , the specificity was 1.00 . Kappa was 0.89 . Considering a gold-standard assessment of age-associated memory impairment as cognitive impairment, the sensitivity of the telephone approach is 0.38  specificity 0.96  and kappa 0.61 .Use of a telephone interview to identify people with dementia or cognitive impairment is a promising and relatively inexpensive strategy for identifying potential participants in intervention and clinical research studies and for classifying subjects in epidemiologic studies. Epidemiologic studies of dementia generally require a large number of subjects. Longitudinal study designs that start with subjects who are cognitively intact and follow them to ascertain the development of dementia are often needed to reduce bias. Although physician and neuropsychological evaluations are considered the gold standard for the diagnosis of dementia, the high cost of these methods makes them infeasible for population-based epidemiological surveys. Administration of mental status screening tests and functional questionnaires by telephone offers a less costly alternative. These methods minimize participant burden, permit standardization and use less costly personnel. However, few studies have examined the reliability and validity of telephone-based methods in correctly identifying cognitive impairment and dementia .This study evaluates whether a brief multi-stage telephone-based cognitive test and functional questionnaire strategy can reliably classify cognitive status when compared to a gold standard in-person clinical assessment. In this study, the gold standard was a classification derived by in-person neurological and neuropsychological evaluation at a university-based Alzheimer Disease Research Center. The staged telephone assessment of cognitive status has been used in the Kaiser Permanente Women's Memory Study, a large ongoing epidemiologic study of dementia in a sample of 3,681 women 75 and older. The same staged model was used in the veteran twin study by Gallo and Breitner  and a siThe study compared the classification of elderly patients into 3 categories  using two different assessment approaches. The gold standard reference classification was performed at the University of Southern California Alzheimer Disease Research Center (USC ADRC) and involved clinical examination of patients by a neurologist and a team experienced in the diagnosis of dementia and cognitive impairment. The second approach relied on information from telephone interviews, the Telephone Interview of Cognitive Status-modified (TICSm) ,5, and aThe subjects in this study were enrolled in a longitudinal study of normal aging, cognitive impairment, and Alzheimer-type dementia at the University of Southern California (USC) Alzheimer Disease Research Center (ADRC). Initial evaluations involve clinical examination of patients by a Board-certified neurologist and a licensed neuropsychologist experienced in the assessment and diagnosis of dementia. The neuropsychological battery was comprised of 20 tests of memory, language, executive control, and construction. In addition, scales of mood and psychiatric symptoms and screenings for visual or auditory impairment were administered to the subject. A rating of functional ability was provided by a collateral informant. See Appendix I for a list of the tests given.Patients evaluated at the USC ADRC are classified as possible or probable AD, mild cognitive impairment (MCI), age-associated memory impairment (AAMI), or normal . One patFollow-up evaluation is comprised of an interval history, depression, behavior and functional questionnaire and neuropsychological testing. Follow-up evaluation is conducted annually for subjects who are classified as cognitively impaired, demented or cognitively intact but over age 80 years and is repeated every 2 years for normal controls less than 80 years old. The results of the follow-up evaluation and neuropsychological testing are reviewed by a team of neurologists and psychologists and the diagnostic classification is updated accordingly. For this study, the most recent classification was used.Ninety-eight subjects  at the USC ADRC during the past 2 years were invited to participate in this study. They were contacted by letter by the USC ADRC (with a return opt-in postcard) to obtain initial permission to contact for a possible telephone interview. Postcard or verbal permission to contact was obtained in 50 cases.Kaiser Permanente investigators mailed an informed consent form to the subject or proxy. Shortly thereafter, a telephone contact was made. Participants read and discussed the consent with the research assistant and were instructed to sign and return the written consent form by mail. If consent was obtained, telephone contact information for these participants (and their consenting proxies for patients with dementia) and appointment times were provided to the survey research firm that conducts the telephone cognitive assessments.Interview responses were reviewed using the procedures described below. Both the telephone interviewers and the experts making the classification of cognitive status based on the TDQ were blinded to the results of the USC ADRC assessment until all subjects in the study had been classified.The first step in the Women's Memory Study assessment of dementia and cognitive status is administration of the 23-question TICSm, which takes from 8 to 15 minutes to administer. The TICSm is strongly correlated with the Mini-Mental State Exam , a more For those classified as possibly impaired based on the TICSm score, an attempt is made to do a second stage cognitive assessment in which the Telephone Dementia Questionnaire (TDQ), is administered to a proxy. The TDQ is a previously validated instrument ,14 that TDQ classification in the dementia category requires memory deficits and multiple impairments in another cognitive domain and at least one deficit in an additional cognitive domain. Classification in the category of "no or minimal impairment" requires no more than two "yes" responses to questions about problems with memory. Classification as cognitive impairment without definite evidence of dementia requires a scatter of deficits that were not sufficient for a dementia classification but were too many to be classified as no dementia. Overall, classification of dementia based on the two-stage telephone classification is "conservative" in that it requires multiple impairments in memory plus 2 additional cognitive domains and functional impairment.The reviewers then make a consensus TDQ classification in the same three categories after discussion of the independent assessments. When there is agreement among the reviewers, no further review is done. When the reviewers disagree, a third trained reviewer, a neuropsychologist or a neurologist, assesses the TDQ responses and makes a final classification. The test reliability of the consensus TDQ has been measured on an on-going basis by having the reviewers reassess TDQs selected at random and blinded to the initial assessment. The kappa coefficient was 0.85 for the consensus assessments.The analysis compared the USC ADRC final diagnosis of cognitive status and the Women's Memory Study classification based on telephone surveys. The unweighted kappa statistic was calculated comparing the ADRC and the staged telephone classification. As this was the principal aim of the Women's Memory Study method, the first comparison included dementia and no dementia classifications only.Other comparisons were based on two alternate assumptions about patients who had been classified by the USC ADRC as having Age-Associated Memory Impairment (AAMI). The first assumption was that these patients were normal and the second was that they had cognitive impairment. Sensitivity for classification with dementia and specificity for classification as normal were also calculated under both sets of assumptions. Ninety-five percent confidence intervals for kappa were estimated as described in Fleiss . ConfideFifty patients who completed the USC ADRC evaluations initially consented to participate in this study. We were able to complete the TICSm assessments on 38 (76%) of these patients. The mean age of participants was 78.1 years (SD ± 8.0) and 26 (68%) were female. Eight of the 38 patients scored below 28 on the TICSm. We then completed TDQ interviews for 8 of the 8 proxies (100%).Table Table Tables Table Our study shows that assessment of dementia status in elderly subjects by a multi-stage telephone method has good agreement with a comprehensive in-person dementia assessment. Sensitivity and specificity for classification of cognitively normal or no dementia were also good. The one case of dementia classified falsely as not impaired in the telephone assessment may have been due to the high educational level (BA degree) and IQ  of this subject. Our approach does not make adjustments for education in the TICSm cutpoint, therefore dementia in persons with high premorbid education and/or IQ may be missed.Note that dementia in this study was confined to AD dementia and the definition of dementia required multiple problems with memory. This study does not specifically address the accuracy of the telephone methods for detecting other types of dementia which are not characterized by predominant memory impairment.On the other hand, sensitivity and specificity of the telephone approach for cognitive impairment was low, especially if one considers AAMI as a form of cognitive impairment. Even in clinical settings, however, the criteria for measuring and defining mild cognitive impairment (MCI) are not uniform or standardized ,19. ConsReliance on the telephone assessment would misclassify some subjects. In general, misclassification in epidemiologic studies causes a bias to the null when it is non-differential . In largEpidemiologic studies that involve in-person assessments of cognition are costly compared with those that rely solely on telephone assessment. We estimate that the telephone strategy used here costs about $38.00 per person assessed when the percentage of those assessed who "fail" the TICSm (score ≤ 27) is 25%. The costs of in-person assessments are generally 20 times that cost.The costs of in-person assessments also affected the number of cases in this analysis. The small number of patients who were willing to be interviewed for the in-person assessments as well as follow-up telephone interviews is a further limitation. In our previous work we have found that there tends to be a bias toward non-participation for the cognitively impaired and demented .Use of the multi-stage telephone strategy to identify people who have dementia or cognitive impairment is promising as a way to identify potential participants in intervention or clinical research. Use of the strategy in dementia detection studies is less costly, does not require representative samples, can be generally smaller than epidemiologic studies, and can be designed to validate eligibility prior to study enrollment.None of the authors have competing personal or financial interests in the interpretation or presentation of this data.VCC & DBP contributed to all aspects of design, analyses and implementation and interpretation of study, and drafts, revisions and critical review of paper.LC & HC contributed to implementation of study, interpretation of data and critical review of paper.VC contributed to analyses and critical review of paper.All have given final approval of this submission.ScreeningMini Mental State ExaminationShort Blessed TestEstimated Premorbid IQAmerican Version of the Nelson Adult Reading Test (AMNART)LanguageBoston Naming TestPhrase repetition – Boston Diagnostic Aphasia ExamOral Word Association TestAnimal NamingToken Test – Multilingual Aphasia ExaminationConstructionRey-Osterrieth Complex Figure, copyJudgment of Line OrientationClock DrawingBlock Design -WAIS- RMemoryLogical Memory I and II- Wechsler Memory Scale IIICalifornia Verbal Learning Test – IIFaces I and II – Wechsler Memory Scale IIIRey-Osterrieth Complex Figure 3' DelayExecutive ControlDigit Span, forward and backward – WAIS-RTrail Making, A and BNumber-Letter Sequencing – Wechsler Memory Scale IIIRepeated patternsEmotional StatusGeriatric Depression ScaleSymptoms Checklist 90-RFunctional AbilityClinical Dementia Rating Scale The pre-publication history for this paper can be accessed here:


Synchronous presentation of more than one germ cell tumours of different histology in the same patient is considered to be very rare. In these cases of multiple germ cell tumours, strong theoretical and clinical data suggest an underlying common pathogenetic mechanism concerning genetic instability or abnormalities during the pluripotent embryonic differentiation and maturation of the germ cell.A 25 year-old young man presented with an enlarging, slightly painful left cervical mass. Despite the initial disorientation of the diagnosis to a possible thyroid disorder, the patient underwent complete surgical resection of the mass revealing mediastinal choriocarcinoma. Subsequent ultrasound of the scrotum indicated the presence of a small lobular node in the upper pole of the left testicle and the patient underwent radical left inguinal orchiectomy disclosing a typical seminoma. Based on these results, the patient received 4 cycles of Bleomycin, Etoposide and Platinum chemotherapy experiencing only mild toxicity and resulting in complete ongoing clinical and biochemical remission.The pathogenesis of concurrent germ cell tumours in the same patient remains an area of controversy. Although the genetic instability of the pluripotent germ cell offers an adequate explanation, the possibility of metastasis from the primary, less differentiated tumour to a distant location as a more mature subtype cannot be excluded. Possible development of a metastatic site of different histology and thus biological behaviour (e.g choriocarcinoma) should be anticipated. Furthermore, urologists, pathologists and medical oncologists should be meticulous in the original pathological diagnosis in these patients, since there is a significant frequency of germ cell tumours with mixed or overlapping histological elements with diverse potential of evolution and differentiation. Synchronous presentation of more than one germ cell tumors (GCTs) in the same patient is considered to be quite rare. It usually involves almost identical histological subtypes, the possibility of metastasis from the one site to another being the most probable explanation . Here we3), thyroid stimulating hormone (TSH), thyreoglobulin and anti-thyroid antibodies- were within normal limits, with the exception of a moderately elevated erythrocyte sedimentation rate (ESR = 44). Subsequent cervical and thoracic CT scan revealed a multilobular mass  probably arising from the left thyroid lobe, with infiltrating features and heterogeneous density with regions of central necrosis and hemorrhage. The mass submerged into the anterior-posterior mediastinum, in proximity with the great vessels of the heart, dislocating the left common carotid artery and the left vagus nerve without infiltrating them . Pre-chemotherapy levels of β-hCG were 93.400 mIU/ml. The patient completed 4 cycles of therapy without experiencing remarkable toxicity (Neutropenia grade I-II according to the NCI-CTC criteria) and is currently (October 2006) asymptomatic, with ongoing complete clinical and biochemical remission according to the RECIST criteria . A schematic presentation of the whole diagnosis and treatment course including β-hCG titer and chest Xray findings is illustrated in Fig. 5A 25-year-old male was presented in October 2004 with a monthly history of a gradually enlarging, painless, left cervical mass. He had no history of prior thyroid disorder or other disease. Clinically the mass was hard in palpation, firmly attached to the surrounding tissues and was located at the anatomic area of the carotide triangle. Thorough physical examination revealed only a marginal hepatomegaly and splenomegaly. Routine blood tests, biochemical markers and thyroid function tests – including triiodothyronine  compared to that of the original location. Thus, medical oncologists, urologists and pathologists should be very suspicious of the original pathological diagnosis in these patients, since there is a significant frequency of GCTs with mixed or overlapping histological elements with diverse potential of evolution and differentiation. Moreover, given the heterogeneity of these tumours, one could emphasize the necessity of obtaining multiple samples from different tumour areas in every case of GCT so as to minimize the risk of missing important particular components of the tumour who could potentially affect the diagnosis, prognosis or even the therapeutic intervention.The presenting symptom of our patient was an enlarging, painless cervical mass, originally thought to be a thyroid node and is considered to be an extremely rare presentation. With the exception of choriocarcinoma, which gives early haematogenous metastases, testicular tumours usually become apparent in their primary location with painless or slightly painful enlargement of the testicle noticed accidentally by the patient himself. Discolo and Dispaquale reported a case of testicular seminoma with cervical lymphadenopathy as the presenting symptom . Costal Germ cell tumours represent a heterogeneous group of malignant cell lines with a variety of frequently overlapping histological pictures or with mixed components suggesting a common "precursor" embryonic cell dysfunction. Histological conversion to a more mature subtype is theoretically possible in a metastatic location with or without therapeutic intervention, as well as synchronous or metachronous development of two different primary germ cell tumours as a result of a common pathogenetic mechanism concerning genetic instability or abnormalities during the pluripotent embryonic germ cell differentiation and maturation.The authors have not any potential conflicts of interest to disclose. G.M. is a European Society of Medical Oncology (ESMO) fellowship recipient for the year 2005–2006.GM conceived of the study, obtained and analysed the data and wrote the manuscript. GP participated in the design of the study and the analysis of the data. ET participated in the design of the study and the writing of the manuscript.The pre-publication history for this paper can be accessed here:


Ensemble attribute profile clustering is a novel, text-based strategy for analyzing a user-defined list of genes and/or proteins. The strategy exploits annotation data present in gene-centered corpora and utilizes ideas from statistical information retrieval to discover and characterize properties shared by subsets of the list. The practical utility of this method is demonstrated by employing it in a retrospective study of two non-overlapping sets of genes defined by a published investigation as markers for normal human breast luminal epithelial cells and myoepithelial cells.Each genetic locus was characterized using a finite set of biological properties and represented as a vector of features indicating attributes associated with the locus (a gene attribute profile). In this study, the vector space models for a pre-defined list of genes were constructed from the Gene Ontology (GO) terms and the Conserved Domain Database (CDD) protein domain terms assigned to the loci by the gene-centered corpus LocusLink. This data set of GO- and CDD-based gene attribute profiles, vectors of binary random variables, was used to estimate multiple finite mixture models and each ensuing model utilized to partition the profiles into clusters. The resultant partitionings were combined using a unanimous voting scheme to produce consensus clusters, sets of profiles that co-occured consistently in the same cluster. Attributes that were important in defining the genes assigned to a consensus cluster were identified. The clusters and their attributes were inspected to ascertain the GO and CDD terms most associated with subsets of genes and in conjunction with external knowledge such as chromosomal location, used to gain functional insights into human breast biology. The 52 luminal epithelial cell markers and 89 myoepithelial cell markers are disjoint sets of genes. Ensemble attribute profile clustering-based analysis indicated that both lists contained groups of genes with the functional properties of membrane receptor biology/signal transduction and nucleic acid binding/transcription. A subset of the luminal markers was associated with metabolic and oxidoreductase activities, whereas a subset of myoepithelial markers was associated with protein hydrolase activity.Given a set of genes and/or proteins associated with a phenomenon, process or system of interest, ensemble attribute profile clustering provides a simple method for collating and sythesizing the annotation data pertaining to them that are present in text-based, gene-centered corpora. The results provide information about properties common and unique to subsets of the list and hence insights into the biology of the problem under investigation. Recent advances in biomedical technologies such as high-throughput molecular profiling provide the ability to identify large numbers of molecules of interest simultaneously. An accessible and straightforward solution to elucidating why a set of genes might be associated with a given phenomenon is manual inspection of the scientific literature and examination of Web-based resources. PubMed  is the mA variety of approaches have been proposed to address the problem of determining the common function(s)specified by a group of genes. The free-text of PubMed abstracts known to be associated with specific genes have been analyzed using concepts from statistical natural language processing (NLP) . FrequeThis work describes ensemble attribute profile clustering, a novel text-based strategy that employs ideas from statistical information retrieval (IR) and annotation data from gene-centered corpora to discover and characterize groups of genes in a list with shared properties. Whereas NLP focuses on the syntax and semantics of text, statistical IR emphasizes vector space models and probabilistic models for document representation, retrieval and analysis ,20. A gei.e., determining homogeneous groups of profiles such that profiles in the same cluster are more similar to each other than they are to ones in other clusters. The problem of discovering groups of genes with similar patterns of attributes can be addressed using methods and principles that have been employed to find groups of transcript profiles with related patterns of expression. Simple probabilistic graphical models, finite mixture models in particular, have been utilized to cluster vector space representations of transcript abundances (gene expression profiles) " (KOG4289) was associated with SELE, PCDHGA12 , and CDH13 ; and (iv) "Cadherin repeat domain" (cd00031) was associated with FAT2, PCDHGA12, and CDH13. Each of these protein domains represents a superfamily of diverse functions, so further studies using a variety of sequence analysis tools will be required to produce fine-grained information about the specific domain subfamily represented in the MYOEPITHELIAL gene list.The output produced by ensemble attribute profile clustering should be viewed as a useful synthesis of the annotations assigned by gene-centered corpora with the results warranting additional examination. For example, of the genes discussed above, CDH13, FAT2, JAG1, PCDHGA12 and SELE are present in MYOEPITHELIAL consensus cluster 1 so a simple interpretation of the functional biology linking them is a connection to membrane biology Table . HoweverAlthough the pathways in which a protein is involved are important properties of a gene, their exclusion from the vector space model formulated here appears to have had minimal impact on the results described above. Inspection of the KEGG pathways assigned to genes in a consensus cluster largely reinforced the observations made by examination of influential GO and CDD attributes. In LUMINAL consensus cluster 3, for example, the GO molecular function term "oxidoreductase activity" was an influential attribute and three of the four genes were assigned the KEGG pathway "Oxidative phosphorylation". For other gene lists, it may be necessary to include KEGG pathway attributes in the gene attribute profiles used as input for consensus clustering. In general, the issue is not only a matter of whether certain properties should be included as attributes in the vector space representation of a gene, but also whether the relevant information is present in and hence can be extracted from text-based, gene-centered corpora.Ensemble attribute profile clustering-based analysis of two disjoint lists of breast tissue-related genes demonstrates the utility of this general purpose strategy for discovering and characterizing groups of genes with similar patterns of features. The approach separates the tasks of computing clusters of profiles and determining attributes important in defining each cluster. Thus, one enhancement would be to replace the finite mixture model used to perform one-way probabilistic clustering with a two-way clustering algorithm that estimates groups of genes and groups of attributes simultaneously . An altSince a particular choice of attributes embodies prior knowledge about the properties of genes deemed to be most important, vector space models can be tailored to emphasize different desired aspects. The advantages of employing GO and CDD terms to build profiles is their capacity to capture a broad range of concepts in structural, molecular and cellular biology, the widespread adoption of terms in these controlled vocabularies to annotate genes, and the ability to extract these descriptors for an arbitrary collection of genes from gene-centered corpora. For the LUMINAL and MYOEPITHELIAL collections, the GO and CDD terms appear sufficient to encapsulate the knowledge that would have been added by incorporation of KEGG pathway-based attributes. However, the GO and CDD vocabularies are unable in general to capture information such as normal versus aberrant phenotypes and aspects related to evolutionary biology. To ameliorate the former limitation, genes could be characterized using terms that were binary random variables indicating association with disease classes such as "immune", "metabolic", "cancer", "cardiovascular", "aging", "development", "infection", "neurodegeneration" and so on. However, the consensus clusters could differ because genes would be grouped on the basis of similar patterns of phenotypic properties rather than the protein structural and functional properties examined in this work.Although GO and CDD terms could be augmented with attributes related to phenotypes, the ensuing profiles would remain partial descriptions of genes. To highlight and illustrate this issue, the inability of GO- and CDD-based profiles to capture knowledge about genomic context was investigated. This topic was selected because of increasing interest in the relationship between co-ordinated gene expression and gene clustering in eukaryotes . The chAttributes related to genomic context could be generated by creating a series of binary variables defining the window within which a gene could occur, for example, cytogenetic location such as "Iq", "1", "2q", and "3p.l3", fixed physical distance such as 10 kbp, and region coinciding with a BAC clone. However, the optimal window size is an open question since too small a bin would result in a large increase in the number of attributes to be added to the vector, too large a bin might be uninformative, and different regions of the genome might require different sizes depending on local factors such as gene density.In both the "bag-of-words" representation of documents used commonly in IR and the "bag-of-attributes" representation of genes considered here, the features in a vector are assumed to be unrelated to each other and the order in which they occur is not significant (exchangeability). However, dependencies amongst and between GO and CDD terms means that this assumption of attribute independence is violated in GO- and CDD-based profiles. The directed acyclic graph underpinning the GO vocabulary defines semantic relationships amongst GO terms so that, for example, the term intracellular is a parent of nucleus. The co-occurrence of protein domains is likely to be non-random because, for example, a nucleic acid binding domain is more likely to occur in conjunction with a nuclease domain than with some other domain. Finally, the GO molecular function terms assigned to a gene are related to the presence of specific protein domains in the encoded protein, for example, DNA binding is associated with zinc finger domains.Despite the limitations in ensemble attribute profile clustering discussed above, analysis of genes associated with two normal human breast tissue cell types using GO- and CDD-based gene attribute profiles demonstrates that the strategy provides a simple but useful method for synthesizing prior biological knowledge and facilitating the extraction of new insights. No specific gene is present in both the list of epithelial cell markers and the list of myoepithelial cell markers . HoweverImproved understanding of the biology of the breast requires integrating data and information from multiple sources as well as studies of both normal and aberrant breast tissue. The two gene lists analyzed here were the outcome of a cDNA microarray study of luminal and myoepithelial cells isolated from primary cultures of reduction mammoplasty specimens . ElsewheA published transcript profiling study of two cell types found in normal human breast tissue and obtained from breast-reduction surgery defined two non-overlapping lists of genes: 76 markers for luminal epithelial cells and 131 markers for myoepithelial cells . Each ofN = 52 and N = 89 LocusIDs ("genes") were designated the LUMINAL and MYOEPITHELIAL collections respectively. The list of human breast tissue-related genes in each collection was analyzed as described below.A set of sequence IDs was mapped to entries in the gene-centered corpus LocusLink   the explicit GO and CDD terms in the gene's LocusLink entry, and (ii) all GO terms that were parents of the explicit GO terms as implied by the structure of the GO directed acyclic graph (DAG). A GO term was discarded if it belonged to the set deemed to be uninformative, namely cellular_component, molecular_function, biological_process, cellular_component unknown, molecular_function unknown, biological_process unknown, obsolete cellular component, obsolete molecular function, obsolete biological process, and the Cellular Component term unlocalized.The gene attribute profiles associated with a collection of n the GO  and CDD n the GO  terms foN gene-specific terms provided the palette of attributes used to characterize genes. In the vector space model representation of a gene considered here, the significance of a feature was quantified by treating each attribute as a binary random variable with "1" signifying that the GO/CDD term was associated with a gene and "0" otherwise (the order of features in the vector was not significant). For a particular gene, the weight of a GO term was "1" if it was an explicit GO term for that gene, or if it was an ancestor in the GO DAG of an explicit GO term. The weight of a CDD term was "1" if it was an explicit CDD term . The weights assigned to attributes depend upon the completeness of information present in the gene-centered corpus used to construct the profiles. Given the limitations of such resources and the evolving nature of biomedical research, a weight of "0" was viewed more as an indicator of an absence of knowledge about association with a gene rather than the presence of definitive information about a lack of association.The non-redundant set of GO and CDD terms formed from the union of N feature vectors produced by the preceding steps were eliminated because the GO or CDD terms were assigned to a small number of genes. For the two gene lists examined here and eight others, a number of attributes were associated with only a few genes (data not shown). In each case, the simple heuristic of retaining attributes assigned to at least three genes was found to provide a parsimonious approach to identifying attributes likely to be informative. If P is the number of features that remain after application of all the preceding feature generation and selection steps, the data set used for subsequent clustering was N P-dimensional vector space representations of genes. For the LUMINAL and MYOEPITHELIAL collections, there were 52 43-dimensional and 89 96-dimensional gene attribute profiles respectively.In the feature generation script described above, some of the features in the N P-dimensional gene attribute profiles was used to estimate a simple probabilistic model, a finite mixture model, where each of the P binary random variables was modelled using a Bernouilli distribution. The specific implementation of finite mixture models utilized here, AutoClass C version 3.3.4 [K that best fit the data. Learning a model from data entailed determining K (model selection) and the probability parameters of each cluster (parameter estimation). AutoClass addressed the combinatorial optimization problem via a search over the space of models and parameters using the Expectation Maximization (EM) algorithm. Starting from a random initialization, the iterative search procedure partitioned the data into clusters (classes) and adjusted the parameters to find their  maximum likelihood estimates. This open-ended procedure was continued until a convergence criterion was met. The profiles were not smoothed to account for unobserved events . AutoClass was run from a series of small high-level Perl scripts that also implemented the cluster combination and report-generation steps described below. The scripts transformed the feature matrix output by the feature generation script into the proper AutoClass input format. The following non-default AutoClass parameter settings were used: max_duration = 10800, max_cycles = 500, start_j_list = 40,45, and force_new_search_p = true. A trained K -class AutoClass model was employed to calculate the probability of a profile having been generated by each of the K clusters.A data set of on 3.3.4 , used a To find a consistent partitioning of the profiles, the results from multiple finite mixture models were integrated to produce consensus clusters. A given clustering algorithm can produce different partitions of the same data set depending upon its assumptions about the data, and choices such as the distance metric in tree-based methods. With AutoClass, different initializations can yield distinct numbers of clusters and assignments of data points to clusters. Combining many classifiers, each a weak learner whose strengths and limitations may differ from the others, can produce a system where the prediction performance of the ensemble is competitive with approaches that focus on estimating a single, good classifier (reviewed in ). HoweveThe simple approach used here was based on four independent finite mixture models estimated using a given data set. For each trained model, a disjoint partitioning of the profiles was produced by assigning each gene to the cluster which maximized its probability (a hard assignment). Consensus clusters were identified using a unanimous voting scheme in which pairs of profiles that co-occurred in all partitionings were placed in the same group by the scripts. To implement this voting algorithm, the problem of finding co-occurring pairs of genes in several different AutoClass runs was restated as the problem of finding consistently-comprised components in different graphs. Given this reinterpretation, a part of the voting algorithm was implemented using the standard C++ Boost Graph library  using PeAll genes in the LUMINAL and MYOEPITHELIAL collections could be equated with a consensus cluster, although this outcome is not guaranteed by the voting algorithm. Although further studies are required to devise methods for selecting the optimal number of partitionings to combine and the voting scheme itself, experiments indicated that the choice of four independent AutoClass models and a unanimous voting scheme yielded consensus clusters that were relatively consistent (data not shown). Increasing the number of partitionings but retaining unanimous voting produced more consensus clusters, each with fewer genes. With a majority voting scheme in which genes needed to co-occur in only three of the four partitionings, the result was consensus clusters whose makeup was largely unchanged together with reassignment of a small number of genes.To elucidate the shared properties of genes in a consensus cluster, attributes important in defining the cluster were determined and the results inspected in conjunction with external knowledge. To assist in interpreting consensus clusters, two complementary reports were generated automatically using a Perl script and the SQL annotation database. The first report gave the annotation counts and relative frequencies of all attributes associated with genes in each consensus cluster. The second report provided a number of other annotations for a gene found in LocusLink: gene symbol, GO and CDD terms, cytoband, and physiological pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database .For the purpose of analysis, a set of "influential" attributes was defined for each consensus class. For a consensus cluster with two or more genes, influential attributes were defined as those attributes assigned to at least half of the genes assigned in that consensus cluster. For a consensus cluster with two genes, influential attributes were those assigned to both genes.The chromosomal positions of genes in the LUMINAL and MYOEPITHELIAL collections were determined as follows. Every LocusID for a gene was mapped to its RefSeq and the RefSeq mapped to chromosomal coordinates in the May 2004 UCSC build of the Golden Path database, specifically, the maximal transcription start and end coordinates in the table refFlat . These cJ.R.S. wrote the software and performed the experiments. A.R. and M.J.B. analyzed the data. I.S.M. conceived the study and analyzed the data. All authors wrote and approved the manuscript.Information on the 52 genes in the LUMINAL collection. For each gene, the "LocusID", "Symbol", "Name", "Cytoband Location", "GO Terms", "Domain Terms", "KEGG Pathway" and "OMIM" fields contain data taken from the July 2004 release of LocusLink. Genes are grouped by their assigned consensus clusters and ordered by their cytoband locations within each cluster. The GO and CDD terms shown are attributes in the gene attribute profiles used as input for probablistic clustering (explicit terms assigned to a gene by LocusLink plus implicit GO terms). The attributes marked with an asterisk are influential attributes, GO/CDD terms that occur with a frequency > 0.5 in clusters with two or more genes.Click here for fileInformation on the GO and CDD attributes associated with the consensus clusters discovered for genes in the LUMINAL collection. For each consensus cluster, the file shows the number of genes assigned to the cluster, along with the absolute count and relative frequency of all attributes associated with the cluster's genes. The section labeled "Summary" shows all the attributes associated with genes in the data set, ordered by average count across all consensus clusters.Click here for fileInformation on the 89 genes in the consensus clusters discovered and characterized for the MYOEPITHELIAL collection, along with their associated GO and CDD attributes. The formats are the same as for luminal-all.html and luminal-attrfreq.html.Click here for fileInformation on the 89 genes in the consensus clusters discovered and characterized for the MYOEPITHELIAL collection, along with their associated GO and CDD attributes. The formats are the same as for luminal-all.html and luminal-attrfreq.html.Click here for fileInformation on the chromosomal locations of the luminal epithelial (brown) and myoepithelial (green) genes. For each gene, the "LocusID" and "Symbol" fields are taken from the July 2004 release of LocusLink. The "Cytoband", "Start" and "End" fields are taken from the May 2004 UCSC assembly of the human genome. Genes are ordered by chromosomal coordinates (p arm followed by q arm). The "Overall" column indicates the distance in kilobases between the adjacent transcription end and start coordinates of the two closest genes from the union of the LUMINAL and MYOEPITHELIAL collections, as well as the number of RefSeqs found in this interval. "pter" and "qter" indicate the number of genes from a telomere to the closest gene in either collection.Click here for file


While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit.Reduced protein stability  The intracellular space presents a highly crowded limited volume predicted to favor association processes -HSQC -HSQC ,19 or ma1H]-HSQC  have beentration  has incrStability of Unpurified proteins from rates of H/D exchange (SUPREX) developed by Oas and coworkers [Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry [in vivo by measuring the hydrogens that exchange with solvent deuterons at a rate depending on the thermodynamic stability of the protein [6–85) estimated with this technique shows no change in stability versus measurements made in vitro [GoN-M) between the native and unfolded states of mutated protein (ΔGoM) compared to the wild-type counterpart (ΔGoN). The bacterial cells are viable up to 3 M urea, which is sufficient to map the entire unfolding transition curves in vivo. Although some successful examples to determine conformational changes and stabilities have been reported so far, both techniques, NMR and mass spectrometry, are far from being established as routine tests for estimating the in vivo stability. A serious limitation arises if the target protein interacts with any cellular components .An interesting twist using conventional mass spectrometry technique, widely applied to the determination of conformational states of proteins, is a method to measure the oworkers . This aptrometry  and dete protein . The SUPin vivo protein stability and folding efficiency, provided the reporter does not change the solubility of the target protein [Fig. in vivo. Davidson and coworkers used the resistance against chloramphenicol generated by fusing chloramphenicol acetyltransferase  to the C-terminus of the protein of interest to select soluble variants from a large pool of mutants created by a random mutagenesis [The fusion of a protein of interest to a reporter protein whose function depends on the solubility of the chimera has been successfully used to reveal agenesis .Kd, which is a significant hurdle to study kinetics of folding/aggregation. An advantage this system shares with the CAT-based system is the potential for using an easily detectable phenotypic selection for screening of mutants with improved folding efficiency.A specific implementation of the reporter-based approach is the structural complementation that involves the splitting of the reporter protein into two components that need to combine to execute their function. Wigley et al.  have desex vivo after the intermolecular association event has taken place. It may perturb the equilibrium established within the cell due to this physical separation of the cellular components. The method is qualitative, allowing the study of alterations of the structure caused by mutations or intracellular environment but it cannot be used for measurements and for determination of the destabilization energy (ΔΔGoN-M) caused by the corresponding mutation.An alternative to the complementarity principle is the split-technique by which target protein is tagged at both termini with two halves of the reporter protein . The proin vivo Förster-resonance-energy-transfer (FRET) measurements of two proteins, reminiscent of the idea of the complementation assay. The target protein is labeled at both termini with GFP and its blue-shifted variant, BFP, and efficient folding brings both fluorescent proteins into sufficient proximity to generate a FRET-signal. The readouts of the fluorescent reporter protein-based methods for protein solubility in vivo are simply detectable signals and allow a fast selection of the soluble variants of any protein of interest. Although enhanced variants of GFP with significantly accelerated folding and fluorescence acquisition have been developed, unfortunately, their bulky size (238 amino acids) may perturb the structure of the target protein. The relatively long folding trajectory of GFP itself ranging from minutes to hours [The use of autofluorescent proteins as reporter protein tags, such as green fluorescent protein (GFP) and its relatives with improved spectral properties, has contributed greatly to our understanding of intracellular trafficking, localization and protein-protein interactions that could not have been achieved with other technologies . The autto hours , even ofto hours .All reporter-based approaches monitor the protein folding and the solubility as a result of efficient folding at its equilibrium. For understanding the mechanisms of protein misfolding in the cell and its impact for disease and biotechnology, it is necessary to follow the process from its early initial steps. Insertion of the reporter molecule within the sequence of the target protein can probably capture early folding/unfolding events. Unfortunately, the bulky reporter proteins can be appended only to either the N- or C-terminus. Short peptide sequences that mediate the labeling of the protein of interest with small synthetic molecules, conferring unique spectroscopic properties, present an alternative solution to overcome these restrictions. Such approaches are discussed in the next section.in vivo labeling of fusion proteins with small synthetic molecules is based on the formation of stable complexes between biarsenical derivatives of fluorescein (FlAsH) or resorufin (ReAsH) and peptides containing tetracysteine motif developed in the lab of Roger Tsien [Kd [The prototype for the er Tsien ,7. The ter Tsien ), which sien [Kd ). The baDye ligation to the tetracysteine receptor is not connected to a characteristic emission shift that can be used as a specific fingerprint for detection of a distinct conformational species. Incorporation of the tetracysteine sequence in flexible structural regions  within the protein sequence of the cellular retinoic acid binding protein, however, leads to a significant sensitivity of the dye quantum yield on the conformational state of the host protein, with a denatured state hyperfluorescent compared to the native one . The FlAin vivo. Nakanishi and coworkers [Recent improvements of the original FlAsH-methodology have expanded the utility of this approach, providing a powerful tool for studying conformational dynamics oworkers  modifiedIn vivo investigations, in the same background the newly synthesized protein encounters, are tremendously helpful to elucidate the mechanisms of protein folding/misfolding. The general need, however, goes towards detecting soluble and insoluble species after the biosynthesis process has reached equilibrium. Besides the development of methods with easy readouts, the approaches have to be conceptually focused on spanning the time window towards the early folding/unfolding events. Real-time kinetic studies are necessary to track the entire process and they will accelerate the development of strategies to suppress the undesired off-pathway unfolding reactions. In addition to understanding the mechanisms of folding and aggregation within the cell, the direct monitoring of the protein solubility in vivo has wide practical applicability. For instance, in the recombinant production of proteins, culture conditions can be modulated and optimized by monitoring their direct effect on the protein solubility. Of particular interest would be to engineer expression hosts, e.g., use of bacterial strains deficient in certain intracellular proteases, or strains with up-regulated expression of cellular chaperones and to follow in real time their influence on the recombinant protein production. The direct monitoring of the folding ability in vivo opens up the possibility of high-throughput screening for the production of proteins with improved solubility by random mutagenesis and facilitating the design of novel protein structures.Off-pathway misfolding and aggregation are highly undesirable processes both reflecting the cell's fitness, resulting in physiological dysfunctions , and the production of recombinant proteins.