Ultrasound imaging links soleus muscle neuromechanics and energetics during human walking with elastic ankle exoskeletons.

Unpowered exoskeletons with springs in parallel to human plantar flexor muscle-tendons can reduce the metabolic cost of walking. We used ultrasound imaging to look 'under the skin' and measure how exoskeleton stiffness alters soleus muscle contractile dynamics and shapes the user's metabolic rate during walking. Eleven participants (4F, 7M; age: 27.7 ± 3.3 years) walked on a treadmill at 1.25 m s-1 and 0% grade with elastic ankle exoskeletons (rotational stiffness: 0-250 Nm rad-1) in one training and two testing days. Metabolic savings were maximized (4.2%) at a stiffness of 50 Nm rad-1. As exoskeleton stiffness increased, the soleus muscle operated at longer lengths and improved economy (force/activation) during early stance, but this benefit was offset by faster shortening velocity and poorer economy in late stance. Changes in soleus activation rate correlated with changes in users' metabolic rate (p = 0.038, R2 = 0.44), highlighting a crucial link between muscle neuromechanics and exoskeleton performance; perhaps informing future 'muscle-in-the loop' exoskeleton controllers designed to steer contractile dynamics toward more economical force production.

Quantifying center of pressure variability in chondrodystrophoid dogs.

The center of pressure (COP) position reflects a combination of proprioceptive, motor and mechanical function. As such, it can be used to quantify and characterize neurologic dysfunction. The aim of this study was to describe and quantify the movement of COP and its variability in healthy chondrodystrophoid dogs while walking to provide a baseline for comparison to dogs with spinal cord injury due to acute intervertebral disc herniations. Fifteen healthy adult chondrodystrophoid dogs were walked on an instrumented treadmill that recorded the location of each dog's COP as it walked. Center of pressure (COP) was referenced from an anatomical marker on the dogs' back. The root mean squared (RMS) values of changes in COP location in the sagittal (y) and horizontal (x) directions were calculated to determine the range of COP variability. Three dogs would not walk on the treadmill. One dog was too small to collect interpretable data. From the remaining 11 dogs, 206 trials were analyzed. Mean RMS for change in COPx per trial was 0.0138 (standard deviation, SD 0.0047) and for COPy was 0.0185 (SD 0.0071). Walking speed but not limb length had a significant effect on COP RMS. Repeat measurements in six dogs had high test retest consistency in the x and fair consistency in the y direction. In conclusion, COP variability can be measured consistently in dogs, and a range of COP variability for normal chondrodystrophoid dogs has been determined to provide a baseline for future studies on dogs with spinal cord injury.

Muscle-spring dynamics in time-limited, elastic movements.

Muscle contractions that load in-series springs with slow speed over a long duration do maximal work and store the most elastic energy. However, time constraints, such as those experienced during escape and predation behaviours, may prevent animals from achieving maximal force capacity from their muscles during spring-loading. Here, we ask whether animals that have limited time for elastic energy storage operate with springs that are tuned to submaximal force production. To answer this question, we used a dynamic model of a muscle-spring system undergoing a fixed-end contraction, with parameters from a time-limited spring-loader (bullfrog: Lithobates catesbeiana) and a non-time-limited spring-loader (grasshopper: Schistocerca gregaria). We found that when muscles have less time to contract, stored elastic energy is maximized with lower spring stiffness (quantified as spring constant). The spring stiffness measured in bullfrog tendons permitted less elastic energy storage than was predicted by a modelled, maximal muscle contraction. However, when muscle contractions were modelled using biologically relevant loading times for bullfrog jumps (50 ms), tendon stiffness actually maximized elastic energy storage. In contrast, grasshoppers, which are not time limited, exhibited spring stiffness that maximized elastic energy storage when modelled with a maximal muscle contraction. These findings demonstrate the significance of evolutionary variation in tendon and apodeme properties to realistic jumping contexts as well as the importance of considering the effect of muscle dynamics and behavioural constraints on energy storage in muscle-spring systems.

Young adults with cystic fibrosis have altered trabecular microstructure by ITS-based morphological analysis.

UNLABELLED: Young adults with cystic fibrosis have compromised plate-like trabecular microstructure, altered axial alignment of trabeculae, and reduced connectivity between trabeculae that may contribute to the reduced bone strength and increased fracture risk observed in this patient population. INTRODUCTION: The risk of fracture is increased in patients with cystic fibrosis (CF). Individual trabecular segmentation (ITS)-based morphological analysis of high-resolution peripheral quantitative computed tomography (HR-pQCT) images segments trabecular bone into individual plates and rods of different alignment and connectivity, which are important determinants of trabecular bone strength. We sought to determine whether alterations in ITS variables are present in patients with CF and may help explain their increased fracture risk. METHODS: Thirty patients with CF ages 18-40 years underwent DXA scans of the hip and spine and HR-pQCT scans of the radius and tibia with further assessment of trabecular microstructure by ITS. These CF patients were compared with 60 healthy controls matched for age (±2 years), race, and gender. RESULTS: Plate volume fraction, thickness, and density as well as plate-plate and plate-rod connectivity were reduced, and axial alignment of trabeculae was lower in subjects with CF at both the radius and the tibia (p < 0.05 for all). At the radius, adjustment for BMI eliminated most of these differences. At the tibia, however, reductions in plate volume fraction and number, axially aligned trabeculae, and plate-plate connectivity remained significant after adjustment for BMI alone and for BMI and aBMD (p < 0.05 for all). CONCLUSIONS: Young adults with CF have compromised plate-like and axially aligned trabecular morphology and reduced connectivity between trabeculae. ITS analysis provides unique information about bone integrity, and these trabecular deficits may help explain the increased fracture risk in adults with CF not accounted for by BMD and/or traditional bone microarchitecture measurements.

Acute systemic LPS-mediated inflammation induces lasting changes in mouse cortical neuromodulation and behavior.

Systemic lipopolysaccharide (LPS) is widely used to induce a neuroinflammatory response that is associated with short-term 'sickness'-behavior that can include fever, loss of activity, loss of appetite, impaired cognition, anxiety and depression. If large enough or left unchecked, this neuroinflammatory response can become self-perpetuating and lead to long-term neurodegenerative processes. In this study, we assess the longer-term effects of a single systemic LPS injection on electrophysiological neuromodulator effects and basic behavioral analysis in mice. Five months after LPS injection, we find a mild reduction in cortical inhibition and altered temporal dynamics of acetylcholine but not norepinephrine or serotonin neuromodulator effects. Consistent with electrophysiological findings, LPS treated mice showed a deficit in memory performance in the novel object recognition test with no effect on measures of anxiety or despair as measured in the open field test and tail suspension test, respectively. Furthermore, LPS-treated mice showed an increase in acetylcholinesterase activity. As increased acetylcholinesterase activity is associated with reduced acetylcholine signaling and impaired cognitive ability, these studies demonstrate the potential for a single inflammatory event to initiate processes that may lead to long-term neurodegeneration.

Sub-clinical necrotic enteritis in broiler chickens: novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue.

The present study revealed several previously not recognized etiological details in the development of necrotic enteritis (NE) in broilers. We provide evidence that the pathological process leading to mucosal epithelium necrosis follows morphologically distinct phases commencing at the basal domain of the mucosal epithelium and then progressively invading the entire lamina propria. Initially mucosal epithelium appears normal, but as the pathological changes progress throughout the lamina propria, the adjacent enterocytes begin to show features of necrotic cell death and the necrotic process of the epithelium progresses from being focal to locally extensive. Ultra-structural examination showed that primary changes occur at the level of basal and lateral domains of the enterocytes, whereas the apical domain of enterocytes remains intact even in the face of advanced necrotic changes. This indicates that the mucosal necrosis does not result from direct damage to the mucosal epithelium. Rather, the necrotic death of enterocytes is a consequential effect of the destruction of lamina propria, the extra-cellular matrix, and intercellular junctions. The nature of these morphological changes indicates that initiation of the pathological process leading to NE involves proteolytic factors affecting the extra-cellular matrix and cellular junctions. Further studies revealed that, indeed, the elevated activity of collagenolytic enzymes in the mucosal milieu and in intestinal tissue represents an integral component of the pathological process leading to NE. In the first instance we discovered that Clostridium perfringens strains isolated from field cases of NE secrete several potent collagenolytic enzymes. In the second instance we observed that, in comparison to controls, broilers challenged with C. perfringens isolated from field cases of NE show high levels of several collagenolytic enzymes in the intestinal tissue. A major component of the overall collagenolytic activity detected in the intestinal tissue was identified by zymography as matrix metalloproteinases (MMPs). Dominant activity was associated with MMP-2. We confirmed using immuno-histochemistry that this enzyme is expressed at high levels in mucosal tissue showing signs of NE. The high levels of collagenolytic activities, in particular associated with MMP-2, demonstrated in our studies are consistent with the nature of morphological changes observed primarily in extra-cellular matrix (ECM) at the basal domain of enterocytes, as well lateral domains of enterocytes. The lack of changes at the level of apical domain of mucosal epithelium indicates that the lipolytic aspect of alpha toxin in NE is not an essential factor in primary lesions development. Taken together, our findings indicate that the early lesions leading to NE are associated with virulence factors that induce proteolytic activity, rather than lipolytic activity.

Necrotising pneumonia is an increasingly detected complication of pneumonia in children.

Necrotising pneumonia (NP) is a severe complication of community-acquired pneumonia characterised by liquefaction and cavitation of lung tissue. The present study describes the epidemiology, aetiology, management and outcomes of children hospitalised with NP over a 15-yr period. A retrospective observational study of NP cases was conducted from January 1990 to February 2005 analysing clinical presentation, laboratory data, hospital course and long-term follow-up. A total of 80 NP cases were identified, with the number of detected cases increasing from 12, in the period 1993-1996, to 40 in the period 2001-2004. In total, 69 (86%) cases had pleural effusion with a low pH (mean 7.08) and 38 (48%) patients had positive cultures, with Streptococcus pneumoniae as the predominant organism. Recently, other organisms, most notably methicillin-resistant Staphylococcus aureus, emerged. Patients had prolonged hospitalisations (median 12 days). A total of 69 patients required pleural interventions and those receiving chest drainage alone had similar outcomes to those managed surgically. All patients had full clinical resolution within 2 months of presentation. Necrotising pneumonia has increasingly been identified as a complication of paediatric pneumonia. Streptococcus pneumoniae remains the predominant organism, but since 2002, different bacteria have been isolated and the age range of cases has broadened. Despite the serious morbidity, massive parenchymal damage and prolonged hospitalisations, long-term outcome following necrotising pneumonia is excellent.

Inhibition of matrix metalloproteinases prevents peroxynitrite-induced contractile dysfunction in the isolated cardiac myocyte.

BACKGROUND AND PURPOSE: The potent oxidant peroxynitrite (ONOO(-)) induces mechanical dysfunction in the intact heart in part through activation of matrix metalloproteinase-2 (MMP-2). This effect may be independent of the proteolytic actions of MMPs on extracellular matrix proteins. The purpose of this study was to examine the effects of ONOO(-) on contractile function at the level of the single cardiac myocyte and whether this includes the action of MMPs. EXPERIMENTAL APPROACH: Freshly isolated ventricular myocytes from adult rats were superfused with Krebs-Henseleit buffer at 21 degrees C and paced at 0.5 Hz. Contractility was measured using a video edge-detector. ONOO(-) or decomposed ONOO(-) (vehicle control) were co-infused over 40 min to evaluate the contraction cease time (CCT). The effects of ONOO(-) on intracellular [Ca(2+)] were determined in myocytes loaded with calcium green-1 AM. MMP-2 activity was measured by gelatin zymography. KEY RESULTS: ONOO(-) (30-600 microM) caused a concentration-dependent reduction in CCT. Myocytes subjected to 300 microM ONOO(-) had a shorter CCT than decomposed ONOO(-) (14.9+1.5 vs 32.2+3.5 min, n=7-8; P<0.05) and showed increased MMP-2 activity. The MMP inhibitors doxycycline (100 microM) or PD 166793 (2 microM) reduced the decline in CCT induced by 300 microM ONOO(-). ONOO(-) caused shorter calcium transient cease time and significant alterations in intracellular [Ca(2+)] homoeostasis which were partially prevented by doxycycline. CONCLUSIONS AND IMPLICATIONS: This is the first demonstration that inhibition of MMPs protects the cardiac myocyte from ONOO(-)-induced contractile failure via an action unrelated to proteolysis of extracellular matrix proteins.

Inhaled nitric oxide inhibits the release of matrix metalloproteinase-2, but not platelet activation, during extracorporeal membrane oxygenation in adult rabbits.

BACKGROUND/PURPOSE: In neonates receiving extracorporeal membrane oxygenation (ECMO), platelet activation and dysfunction occur with the release of matrix metalloproteinase (MMP)-2, which stimulates platelet aggregation. Because inhaled nitric oxide (NO) reduces pulmonary hypertension and inhibits platelet aggregation, the authors examined the effects of inhaled NO on platelet activation induced by ECMO. METHODS: Ten adult white New Zealand rabbits were instrumented for ECMO and assigned randomly to receive either inhaled NO at 40 ppm or 30% oxygen for 1 hour before ECMO and continued for 4 hours after starting ECMO. Platelet counts, collagen-induced platelet aggregation ex vivo, plasma MMP-2, and MMP-9 activities were measured. RESULTS: (1) ECMO caused thrombocytopenia, decreased platelet aggregation, and increased plasma MMP-2 and MMP-9 activities in controls. (2) Inhaled NO inhibited platelet aggregation before ECMO but did not affect the ECMO-induced thrombocytopenia and platelet activation. (3) Inhaled NO significantly abolished the ECMO-induced increase in plasma MMP-2 but not MMP-9 activities. CONCLUSIONS: Although inhaled NO did not inhibit the platelet activation during ECMO in adult rabbits, it attenuated the increase in plasma MMP-2 activity that may be important for neonates treated with ECMO.

Strains of [PSI(+)] are distinguished by their efficiencies of prion-mediated conformational conversion.

Yeast prions are protein-based genetic elements that produce phenotypes through self-perpetuating changes in protein conformation. For the prion [PSI(+)] this protein is Sup35, which is comprised of a prion-determining region (NM) fused to a translational termination region. [PSI(+)] strains (variants) with different heritable translational termination defects (weak or strong) can exist in the same genetic background. [PSI(+)] variants are reminiscent of mammalian prion strains, which can be passaged in the same mouse strain yet have different disease latencies and brain pathologies. We found that [PSI(+)] variants contain different ratios of Sup35 in the prion and non-prion state that correlate with different translation termination efficiencies. Indeed, the partially purified prion form of Sup35 from a strong [PSI(+)] variant converted purified NM much more efficiently than that of several weak variants. However, this difference was lost in a second round of conversion in vitro. Thus, [PSI(+)] variants result from differences in the efficiency of prion-mediated conversion, and the maintenance of [PSI(+)] variants involves more than nucleated conformational conversion (templating) to NM alone.

Cardiac surgery increases the activity of matrix metalloproteinases and nitric oxide synthase in human hearts.

OBJECTIVES: Heart function is variably impaired after cardiopulmonary bypass. We hypothesized that, similar to other myocardial injury states, cardiopulmonary bypass leads to enhanced activity of nitric oxide synthase and matrix metalloproteinases. METHODS: We obtained right atrial biopsy specimens and plasma samples at the onset and termination of cardiopulmonary bypass in 10 patients. Biopsy specimens were analyzed for nitric oxide synthase activity by using a citrulline assay, whereas plasma and tissue were analyzed for matrix metalloproteinase-9 and matrix metalloproteinase-2 activity by using zymography. Tissue inhibitor of metalloproteinase-4 was analyzed by means of Western blotting. The cellular expression of inducible nitric oxide, endothelial nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 was determined in right atrial biopsy samples from 3 additional patients by using the appropriate conjugated antibodies. RESULTS: Nitric oxide synthase activity increased from the beginning to the end of bypass (4.46 +/- 1.07 vs 16.77 +/- 4.86 pmol citrulline/mg of protein per minute, respectively; P =.018). Pro-matrix metalloproteinase-9 activity increased in hearts (199 +/- 41 vs 660 +/- 177 density units/mg protein; P =.008) and plasma (14.1 +/- 4.6 vs 52.2 +/- 5.9 density units/mg protein; P =.008). Pro-matrix metalloproteinase-2 activity increased in the heart (201 +/- 23 vs 310 +/- 35 density units/mg protein, P <.05) but not in plasma. Tissue inhibitor of metalloproteinase-4 expression in the heart decreased (1574 +/- 280 vs 864 +/- 153 density units, P =.014). CONCLUSIONS: Cardiopulmonary bypass activates enzymes mediating acute inflammation and organ injury (ie, nitric oxide synthase, matrix metalloproteinase-9, and matrix metalloproteinase-2). Decreased tissue inhibitor of metalloproteinase-4 expression allows relatively unopposed increases in matrix metalloproteinase tissue activity. We postulate that these changes play a role in the pathogenesis of heart dysfunction after bypass surgery.

Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast.

Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.

Ultrastructural and molecular changes in the left and right ventricular myocardium associated with ascites syndrome in broiler chickens raised at low altitude.

The present study examines ultrastructural and molecular changes in ventricular myocardium associated with ascites cases in fast-growing broilers raised at low altitude. Extensive ultrastructural lesions were seen in the left and right ventricular myocardium of broilers with fulminant heart failure and ascites. Significant changes included lesions in the myofibril contractile apparatus, altered mitochondria, marked reduction in the myofibril component, and changes in the extracellular matrix (ECM) architecture. No lesions were observed in hearts of slow growing broilers, but mild to moderate changes (predominantly in the left ventriculum) were apparent in the hearts from some clinically normal, fast-growing broilers. SDS-PAGE profiles of washed myofibrils showed several distinctly different bands in preparations from left ventricular myocardium of ascitic birds. Western blot analysis of these samples revealed several fragments of myosin heavy chain, M-protein, and titin. Based on gelatinolytic activity, matrix metalloproteinases (MMP) in the cytosolic fraction of ventricular myocardium homogenates were identified as MMP-2. The relative activity of this enzyme appears to be considerably higher in preparations from broilers, particularly in the preparations from the left ventriculum of fast-growing broilers, in comparison to leghorns or slow growing broilers. The nature and distribution of the changes in the heart indicate that chronic cardiomyopathic process in the left ventricular myocardium occurs during the development of ascites. It is postulated that progressive deterioration of the left heart pump function caused by initial lesions in the left ventricular myocardium is a significant factor in the development of pulmonary hypertension and the pathogenesis of ascites in broilers raised at low altitude.

Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: regulation by nitric oxide.

A correlation exists between the ability of tumor cells to aggregate platelets and their tendency to metastasize. Tumor cell-induced platelet aggregation (TCIPA) facilitates the embolization of the vasculature with tumor cells and the formation of metastatic foci. It is well documented that matrix metalloproteinases (MMPs) play an integral part in tumor spread and the metastatic cascade. Therefore, we have examined the role of MMPs during TCIPA and its regulation by nitric oxide (NO) in vitro. Human HT-1080 fibrosarcoma and A549 lung epithelial cancer cells induced TCIPA in a concentration-dependent manner that was monitored by aggregometry. This aggregation resulted in the release of MMIP-2 from platelets and cancer cells, as measured by zymography. HT-1080 cells released significantly more MMP-2 than A549 cells and were more efficacious in inducing TCIPA. Inhibition of MMP-2 with phenanthroline (1-1000 microM), a synthetic inhibitor of MMPs, and by neutralizing anti-MMIP-2 antibody (10 microg/ml) reduced TCIPA induced by HT-1080 cells. TCIPA was abolished by simultaneous inhibition of platelet function with acetylsalicylic acid (100 microM; thromboxane pathway inhibitor), apyrase (250 microg/ml; ADP pathway inhibitor), and phenanthroline. NO donors such as S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione (both at 0.01-100 microM) inhibited TCIPA and MMP-2 release from platelets and tumor cells. The inhibitory actions of S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione were reversed by 1H-[1,2,4]oxadiazole[4,3]quinoxalin-1-one (0.01-30 microM), a selective inhibitor of the soluble guanylyl cyclase. We conclude that (a) human fibrosarcoma cells aggregate platelets via mechanism(s) that are mediated, in part, by MMP-2; (b) NO inhibits TCIPA, in part, by attenuating the release of MMP-2; and (c) these effects of NO are cGMP-dependent.

Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles.

An effective in vitro model of the placental villous syncytium cultured on semi-permeable substrata is essential for studies of infectious pathogen transmission from mother to fetus. Current models using amniotic membranes or thinner artificial membranes show significant leakage, suggesting disruption of tight junctions or the presence of gaps between syncytial units. Such disruption and discontinuity of trophoblast cultures are probably the result of high stromal cell contamination, poor viability and lack of proliferation in culture. We have successfully cultured confluent layers of tight-junctioned syncytium on semi-permeable insert membranes using highly viable purified cytotrophoblasts and an alternating multiple seeding and differentiation technique. Using criteria including transepithelial diffusion of high and low molecular weight substances, electrical resistance and directional secretion of the matrix metalloproteinase, MMP-9, we demonstrate that these cultures form effective and functional physical barriers that can be maintained for up to 1 month.

Differential expression of matrix metalloproteinases and the tissue inhibitor in human milk.

There are differences in the temporal expression of MMP-2 and TIMP-4 in human milk from Day 1 to 30 postpartum in healthy mothers of term pregnancies: (i) MMP-2 activity peaked at Day 1 (colostrum) then exponentially decreased afterwards; and (ii) The expression of TIMP-4 was maximal at Day 7 and persisted thereafter. From our preliminary findings, the differential expression of MMP and the tissue inhibitor in human milk may be related to the protective action of human milk.

Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts.

The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.

Nucleated conformational conversion and the replication of conformational information by a prion determinant.

Prion proteins can serve as genetic elements by adopting distinct physical and functional states that are self-perpetuating and heritable. The critical region of one prion protein, Sup35, is initially unstructured in solution and then forms self-seeded amyloid fibers. We examined in vitro the mechanism by which this state is attained and replicated. Structurally fluid oligomeric complexes appear to be crucial intermediates in de novo amyloid nucleus formation. Rapid assembly ensues when these complexes conformationally convert upon association with nuclei. This model for replicating protein-based genetic information, nucleated conformational conversion, may be applicable to other protein assembly processes.