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Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.

Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.

Prion diseases are a group of inevitably fatal neurodegenerative disorders affecting numerous mammalian species, including Sapiens. Prions are composed of PrP Sc , the disease specific conformation of the host encoded prion protein. Prion strains are operationally defined as a heritable phenotype of disease under controlled transmission conditions. Treatment of rodents with anti-prion drugs results in the emergence of drug-resistant prion strains and suggest that prion strains are comprised of a dominant strain and substrains. While much experimental evidence is consistent with this hypothesis, direct observation of substrains has not been observed. Here we show that replication of the dominant strain is required for suppression of a substrain. Based on this observation we reasoned that selective reduction of the dominant strain may allow for emergence of substrains. Using a combination of biochemical methods to selectively reduce drowsy (DY) PrP Sc from biologically-cloned DY transmissible mink encephalopathy (TME)-infected brain resulted in the emergence of strains with different properties than DY TME. The selection methods did not occur during prion formation, suggesting the substrains identified preexisted in the DY TME-infected brain. We show that DY TME is biologically stable, even under conditions of serial passage at high titer that can lead to strain breakdown. Substrains therefore can exist under conditions where the dominant strain does not allow for substrain emergence suggesting that substrains are a common feature of prions. This observation has mechanistic implications for prion strain evolution, drug resistance and interspecies transmission.

Prions from cattle with bovine spongiform encephalopathy have transmitted to humans, whereas scrapie from sheep and goats likely has not, suggesting that some prions can cross species barriers more easily than others. Prions are composed of a dominant strain and minor strains, and the contribution of each population to adapt to new replicative environments is unknown. Recently, minor prion strains were isolated from the biologically cloned prion strain DY TME, and these minor prion strains differed in properties from the dominant prion strain, DY TME. Here we found that these minor prion strains also differed in conversion efficiency and host range compared to the dominant strain DY TME. These novel findings provide evidence that minor prion strains contribute to interspecies transmission, underscoring the significance of minor strain components in important biological processes.

Prion diseases are caused by a misfolded isoform of the prion protein, PrPSc. Prion strains are hypothesized to be encoded by strain-specific conformations of PrPSc and prions can interfere with each other when a long-incubation period strain (i.e. blocking strain) inhibits the conversion of a short-incubation period strain (i.e. non-blocking). Prion strain interference influences prion strain dynamics and the emergence of a strain from a mixture; however, it is unknown if two long-incubation period strains can interfere with each other. Here, we show that co-infection of animals with combinations of long-incubation period strains failed to identify evidence of strain interference. To exclude the possibility that this inability of strains to interfere in vivo was due to a failure to infect common populations of neurons we used protein misfolding cyclic amplification strain interference (PMCAsi). Consistent with the animal bioassay studies, PMCAsi indicated that both co-infecting strains were amplifying independently, suggesting that the lack of strain interference is not due to a failure to target the same cells but is an inherent property of the strains involved. Importantly PMCA reactions seeded with long incubation-period strains contained relatively higher levels of remaining PrPC compared to reactions seeded with a short-incubation period strain. Mechanistically, we hypothesize the abundance of PrPC is not limiting in vivo or in vitro resulting in prion strains with relatively low prion conversion efficiency to amplify independently. Overall, this observation changes the paradigm of the interactions of prion strains and has implications for interspecies transmission and emergence of prion strains from a mixture.

Prion diseases are caused by the disease-specific self-templating infectious conformation of the host-encoded prion protein, PrPSc. Prion strains are operationally defined as a heritable phenotype of disease under controlled conditions. One of the hallmark phenotypes of prion strain diversity is tropism within and between tissues. A defining feature of prion strains is the regional distribution of PrPSc in the CNS. Additionally, in both natural and experimental prion disease, stark differences in the tropism of prions in secondary lymphoreticular system tissues occur. The mechanism underlying prion tropism is unknown; however, several possible hypotheses have been proposed. Clinical target areas are prion strain-specific populations of neurons within the CNS that are susceptible to neurodegeneration following the replication of prions past a toxic threshold. Alternatively, the switch from a replicative to toxic form of PrPSc may drive prion tropism. The normal form of the prion protein, PrPC, is required for prion formation. More recent evidence suggests that it can mediate prion and prion-like disease neurodegeneration. In vitro systems for prion formation have indicated that cellular cofactors contribute to prion formation. Since these cofactors can be strain specific, this has led to the hypothesis that the distribution of prion formation cofactors can influence prion tropism. Overall, there is evidence to support several mechanisms of prion strain tropism; however, a unified theory has yet to emerge.

Synthetic prions, generated de novo from minimal, non-infectious components, cause bona fide prion disease in animals. Transmission of synthetic prions to hosts expressing syngeneic PrP C results in extended, variable incubation periods and incomplete attack rates. In contrast, murine synthetic prions (MSP) generated via PMCA with minimal cofactors readily infected mice and hamsters and rapidly adapted to both species. To investigate if hamster synthetic prions (HSP) generated under the same conditions as the MSP are also highly infectious, we inoculated hamsters with HSP generated with either hamster wild type or mutant (ΔG54, ΔG54/M139I, M139I/I205M) recombinant PrP. None of the inoculated hamsters developed clinical signs of prion disease, however, brain homogenate from HSP WT - and HSP ΔG54 -infected hamsters contained PrP Sc , indicating subclinical infection. Serial passage in hamsters resulted in clinical disease at second passage accompanied by changes in incubation period and PrP Sc conformational stability between second and third passage. These data suggest the HSP, in contrast to the MSP, are not comprised of PrP Sc , and instead generate authentic PrP Sc via deformed templating. Differences in infectivity between the MSP and HSP suggest that, under similar generation conditions, the amino acid sequence of PrP influences generation of authentic PrP Sc .

Prion diseases are transmissible protein misfolding disorders that occur in animals and humans where the endogenous prion protein, PrPC, undergoes a conformational change into self-templating aggregates termed PrPSc. Formation of PrPSc in the central nervous system (CNS) leads to gliosis, spongiosis, and cellular dysfunction that ultimately results in the death of the host. The spread of prions from peripheral inoculation sites to CNS structures occurs through neuroanatomical networks. While it has been established that endogenous PrPC is necessary for prion formation, and that the rate of prion spread is consistent with slow axonal transport, the mechanistic details of PrPSc transport remain elusive. Current research endeavors are primarily focused on the cellular mechanisms of prion transport associated with axons. This includes elucidating specific cell types involved, subcellular machinery, and potential cofactors present during this process.

Prion diseases, including chronic wasting disease (CWD), are caused by prions, which are misfolded aggregates of normal cellular prion protein. Prions possess many characteristics that distinguish them from conventional pathogens, in particular, an extraordinary recalcitrance to inactivation and a propensity to avidly bind to surfaces. In middle to late stages of CWD, prions begin accumulating in cervid muscle tissues. Those features collectively create scenarios in which occupational hazards arise for workers processing venison and pose risks to consumers through direct prion exposure through ingestion and cross-contamination of food products. In this study, we demonstrate that steel and plastic surfaces used in venison processing can be directly contaminated with CWD prions and that cross-contamination of CWD-negative venison can occur from equipment that had previously been used with CWD-positive venison. We also show that several decontaminant solutions (commercial bleach and potassium peroxymonosulfate) are efficacious for prion inactivation on those same surfaces.