Methylation at CpG sites related to growth differentiation factor-15 was not prospectively associated with cardiovascular death in discordant monozygotic twins.

Myocardial infarction patients had decreased methylation at four growth differentiating factor-15 (GDF-15) related CpG sites (cg13033858, cg16936953, cg17150809, and cg18608055). These sites had not been studied for their association with cardiovascular disease (CVD) deaths. Thus, we aimed to assess the associations independent of genes, shared environment, and traditional CVD risk factors. Nineteen white, male, monozygotic twin pairs discordant for CVD deaths were included from the National Heart, Lung and Blood Institute Twin Study (NHLBI) initiated in 1969. Data on vital status was collected through December 31, 2014. Methylation of buffy coat DNA at exam 3 (1986-87) was measured using the Illumina HumanMethylation450 BeadChip. Principal component analysis was used to generate a score representing blood leukocyte composition and baseline CVD risk factors and predominated with natural killer cells, CD4+ T cells, and Framingham risk score. Conditional logistic regression demonstrated that methylation at the four CpG sites was not associated with CVD deaths before (all p > 0.05, bootstrapped p > 0.05) and after adjustment for the score (all p > 0.05). Joint influences of cg16936953 and the score were statistically significant (p < 0.05). In conclusion, joint influences of methylation at the site cg16936953 and the score are prospectively associated with CVD deaths independent of germline and common environment.ClinicalTrials.gov Identifier for NHLBI Twin Study: NCT00005124.

Quantification of Brucella abortus population structure in a natural host.

Cattle are natural hosts of the intracellular pathogen Brucella abortus, which inflicts a significant burden on the health and reproduction of these important livestock. The primary routes of infection in field settings have been described, but it is not known how the bovine host shapes the structure of B. abortus populations during infection. We utilized a library of uniquely barcoded B. abortus strains to temporally and spatially quantify population structure during colonization of cattle through a natural route of infection. Introducing 108 bacteria from this barcoded library to the conjunctival mucosa resulted in expected levels of local lymph node colonization at a 1-wk time point. We leveraged variance in strain abundance in the library to demonstrate that only 1 in 10,000 brucellae introduced at the site of infection reached a parotid lymph node. Thus, cattle restrict the overwhelming majority of B. abortus introduced via the ocular conjunctiva at this dose. Individual strains were spatially restricted within the host tissue, and the total B. abortus census was dominated by a small number of distinct strains in each lymph node. These results define a bottleneck that B. abortus must traverse to colonize local lymph nodes from the conjunctival mucosa. The data further support a model in which a small number of spatially isolated granulomas founded by unique strains are present at 1 wk postinfection. These experiments demonstrate the power of barcoded transposon tools to quantify infection bottlenecks and to define pathogen population structure in host tissues.

Brucellosis in India: results of a collaborative workshop to define One Health priorities.

Brucellosis is an important zoonosis worldwide. In livestock, it frequently causes chronic disease with reproductive failures that contribute to production losses, and in humans, it causes an often-chronic febrile illness that is frequently underdiagnosed in many low- and middle-income countries, including India. India has one of the largest ruminant populations in the world, and brucellosis is endemic in the country in both humans and animals. In November 2017, the International Livestock Research Institute invited experts from government, national research institutes, universities, and different international organizations to a one-day meeting to set priorities towards a "One Health" control strategy for brucellosis in India. Using a risk prioritization exercise followed by discussions, the meeting agreed on the following priorities: collaboration (transboundary and transdisciplinary); collection of more epidemiological evidence in humans, cattle, and in small ruminants (which have been neglected in past research); Economic impact studies, including cost effectiveness of control programmes; livestock vaccination, including national facilities for securing vaccines for the cattle population; management of infected animals (with the ban on bovine slaughter, alternatives such as sanctuaries must be explored); laboratory capacities and diagnostics (quality must be assured and better rapid tests developed); and increased awareness, making farmers, health workers, and the general public more aware of risks of brucellosis and zoonoses in general. Overall, the meeting participants agreed that brucellosis control will be challenging in India, but with collaboration to address the priority areas listed here, it could be possible.

Characterization of the NLRP1 inflammasome response in bovine species.

Inflammasomes act as sensors of infection or damage to initiate immune responses. While extensively studied in rodents, understanding of livestock inflammasomes is limited. The NLRP1 inflammasome sensor in rodents is activated by Toxoplasma gondii, Bacillus anthracis lethal toxin (LT), and potentially other zoonotic pathogens. LT activates NLRP1 by N-terminal proteolysis, inducing macrophage pyroptosis and a pro-inflammatory cytokine response. In contrast, NLRP1 in macrophages from humans and certain rodent strains is resistant to LT cleavage, and pyroptosis is not induced. Evolution of NLRP1 sequences towards those leading to pyroptosis is of interest in understanding innate immune responses in different hosts. We characterized NLRP1 in cattle (Bos taurus) and American bison (Bison bison). Bovine NLRP1 is not cleaved by LT, and cattle and bison macrophages do not undergo toxin-induced pyroptosis. Additionally, we found a predicted Nlrp1 splicing isoform in cattle macrophages lacking the N-terminal domain. Resistance to LT in bovine and human NLRP1 correlates with evolutionary sequence similarity to rodents. Consistent with LT-resistant rodents, bovine macrophages undergo a slower non-pyroptotic death in the presence of LPS and LT. Overall, our findings support the model that NLRP1 activation by LT requires N-terminal cleavage, and provide novel information on mechanisms underlying immune response diversity.

Teaching Trek: Reflections on Participation in the Indo-U.S. Teaching Professorship Program.

The American Society for Microbiology's Indo-U.S. Teaching Professorship program (sponsored by the Indo-U.S. Science and Technology Forum) provides an opportunity for cross-continental teaching exchanges in the microbial sciences. In this article, I reflect on my experiences as a 2017 Teaching Professor at the University of Delhi, where I developed and delivered a curriculum about prion diseases and a workshop on scientific communication. Tips for preparation, informal assessments, and portable classroom manipulatives are presented for educators who are interested in participating in similar programs. International teaching exchanges provide a unique opportunity to develop skills in assessment and adaptability while meeting new colleagues from across the globe.

Coincidence cloning recovery of Brucella melitensis RNA from goat tissues: advancing the in vivo analysis of pathogen gene expression in brucellosis.

BACKGROUND: Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection. RESULTS: Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph nodes of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of Brucella gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at B. melitensis gene expression patterns in vivo, the subset of Brucella genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction. CONCLUSIONS: Overall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in Brucella persistence in natural host systems.

Riboflavin Riboswitch Regulation: Hands-On Learning about the Role of RNA Structures in the Control of Gene Expression in Bacteria.

American Society for Microbiology (ASM) Curriculum Guidelines highlight the importance of instruction about informational flow in organisms, including regulation of gene expression. However, foundational central dogma concepts and more advanced gene regulatory mechanisms are challenging for undergraduate biology students. To increase student comprehension of these principles, we designed an activity for upper-level biology students centered on construction and analysis of physical models of bacterial riboswitches. Students manipulate an inexpensive bag of supplies (beads, pipe cleaners) to model two conformations of a riboswitch in a bacterial transcript. After initial pilot testing, we implemented the activity in three upper-level classes at one research-intensive and two primarily undergraduate institutions. To assess student perceptions of learning gains, we utilized a pre/post-activity 5-point Likert-type survey instrument to characterize student perceptions of confidence in both their understanding of riboswitches and their ability to apply the central dogma to riboswitches. Median post-test ranks were significantly higher than median pre-test ranks (p < 0.0001) when compared by the Wilcoxon signed-rank test (n = 31). Next, we assessed post-activity knowledge via use of a rubric to score student responses on exam questions. More than 80% of students could correctly describe and diagram examples of riboswitches; data from initial iterations were used to enhance curriculum materials for subsequent implementations. We conclude that this riboswitch activity leads to both student-reported increases in confidence in the ASM curriculum dimension of gene regulation, including central dogma concepts, and demonstrated student ability to diagram riboswitches, predict outcomes of riboswitches, and connect riboswitches to evolutionary roles.

Inflammasomes in livestock and wildlife: Insights into the intersection of pathogens and natural host species.

The inflammasome serves as a mechanism by which the body senses damage or danger. These multiprotein complexes form in the cytosol of myeloid, epithelial and potentially other cell types to drive caspase-1 cleavage and the secretion of the pro-inflammatory cytokines IL-1ß and IL-18. Different types of inflammasomes, centered on (and named after) their cytosolic NLRs, respond to signals from bacteria, fungi, and viruses, as well as "sterile inflammatory" triggers. Despite the large body of research accumulated on rodent and human inflammasomes over the past 15 years, only recently have studies expanded to consider the role of inflammasomes in veterinary and wildlife species. Due to the key role of inflammasomes in mediating inflammatory responses observed in humans and rodents, characterization of the similarities and differences between humans/rodents and veterinary species is required to identify genetic and evolutionary influences on disease responses and to develop therapeutic candidates for use in veterinary inflammatory syndromes. Here, we summarize recent findings on inflammasomes in swine, cattle, dogs, bats, small ruminants, and birds. We describe current gaps in our knowledge and highlight promising areas for future research.

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

Pathologic and biochemical characterization of PrPSc from elk with PRNP polymorphisms at codon 132 after experimental infection with the chronic wasting disease agent.

BACKGROUND: The Rocky Mountain elk (Cervus elaphus nelsoni) prion protein gene (PRNP) is polymorphic at codon 132, with leucine (L132) and methionine (M132) allelic variants present in the population. In elk experimentally inoculated with the chronic wasting disease (CWD) agent, different incubation periods are associated with PRNP genotype: LL132 elk survive the longest, LM132 elk are intermediate, and MM132 elk the shortest. The purpose of this study was to investigate potential mechanisms underlying variations in incubation period in elk of different prion protein genotypes. Elk calves of three PRNP genotypes (n = 2 MM132, n = 2 LM132, n = 4 LL132) were orally inoculated with brain homogenate from elk clinically affected with CWD. RESULTS: Elk with longer incubation periods accumulated relatively less PrPSc in the brain than elk with shorter incubation periods. PrPSc accumulation in LM132 and MM132 elk was primarily neuropil-associated while glial-associated immunoreactivity was prominent in LL132 elk. The fibril stability of PrPSc from MM132 and LM132 elk were similar to each other and less stable than that from LL132 elk. Real-time quaking induced conversion assays (RT-QuIC) revealed differences in the ability of PrPSc seed from elk of different genotypes to convert recombinant 132 M or 132 L substrate. CONCLUSIONS: This study provides further evidence of the importance of PRNP genotype in the pathogenesis of CWD of elk. The longer incubation periods observed in LL132 elk are associated with PrPSc that is more stable and relatively less abundant at the time of clinical disease. The biochemical properties of PrPSc from MM132 and LM132 elk are similar to each other and different to PrPSc from LL132 elk. The shorter incubation periods in MM132 compared to LM132 elk may be the result of genotype-dependent differences in the efficiency of propagation of PrPSc moieties present in the inoculum. A better understanding of the mechanisms by which the polymorphisms at codon 132 in elk PRNP influence disease pathogenesis will help to improve control of CWD in captive and free-ranging elk populations.

Effects of a naturally occurring amino acid substitution in bovine PrP: a model for inherited prion disease in a natural host species.

OBJECTIVE: The most common hereditary prion disease is human Creutzfeldt-Jakob disease (CJD), associated with a mutation in the prion gene resulting in a glutamic acid to lysine substitution at position 200 (E200K) in the prion protein. Models of E200K CJD in transgenic mice have proven interesting but have limitations including inconsistencies in disease presentation, requirement for mixed species chimeric protein constructs, and the relatively short life span and time to disease onset in rodents. These factors limit research on the mechanism by which the mutation drives disease development. Therefore, our objective was to provide the first assessment of cattle carrying the homologous mutation, E211K, as a system for investigating longer-term disease mechanisms. The E211K substitution was associated with a case of bovine spongiform encephalopathy from 2006. RESULTS: We assessed the molecular properties of bovine E211K prion protein, characterized the molecular genetics of a population of cattle E211K carriers (offspring of the original EK211 cow) in relation to findings in humans, and generated preliminary evidence that the impacts of copper-induced oxidative stress may be different in cattle as compared to observations in transgenic mouse models. The cattle E211K system provides the opportunity for future analysis of physiological changes over time.

In vivo characterization of an Hfq protein encoded by the Bacillus anthracis virulence plasmid pXO1.

BACKGROUND: Bacterial Hfq proteins post-transcriptionally regulate gene expression, primarily by mediating the interaction between sRNAs (small RNAs) and their target mRNAs. The role of Hfq-based regulation has been well defined in Gram-negative bacteria, but comparatively less is known about the impact of Hfq proteins in Gram-positive species. The Gram-positive pathogen Bacillus anthracis (causative agent of anthrax) is distinct in that it expresses three homologs of Hfq: Hfq1 and Hfq2 from the chromosome, and Hfq3 from the pXO1 virulence plasmid. RESULTS: In this study, we utilized overexpression as a strategy to examine the impact of Hfq3 on B. anthracis physiology. The increase in Hfq3 protein levels led to anomalous cell shape and chain formation, which manifested as a severe growth defect. This phenotype was specific to B. anthracis, as Hfq3 expression in B. subtilis at similar levels was not toxic. Toxicity was dependent on residues on the distal face of Hfq3 that are involved in mRNA binding in other bacterial species. CONCLUSIONS: Thus, we hypothesize that Hfq3 interacts with RNA(s) involved in essential functions in the B. anthracis cell, leading to increased binding upon overexpression that either sequesters or accelerates degradation of RNAs important for growth. These results not only aid in elucidating the role of Hfq proteins in B. anthracis, but also contribute to our current understanding of Hfq in Gram-positive bacteria.

A Comparison of Classical and H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism in Wild-Type and EK211 Cattle Following Intracranial Inoculation.

In 2006, a case of H-type bovine spongiform encephalopathy (BSE-H) was diagnosed in a cow that was associated with a heritable polymorphism in the bovine prion protein gene (PRNP) resulting in a lysine for glutamate amino acid substitution at codon 211 (called E211K) of the prion protein. Although the prevalence of this polymorphism is low, cattle carrying the K211 allele may be predisposed to rapid onset of BSE-H when exposed or to the potential development of a genetic BSE. This study was conducted to better understand the relationship between the K211 polymorphism and its effect on BSE phenotype. BSE-H from the US 2006 case was inoculated intracranially (IC) in one PRNP wild-type (EE211) calf and one EK211 calf. In addition, one wild-type calf and one EK211 calf were inoculated IC with brain homogenate from a US 2003 classical BSE case. All cattle developed clinical disease. The survival time of the E211K BSE-H inoculated EK211 calf (10 months) was shorter than the wild-type calf (18 months). This genotype effect was not observed in classical BSE inoculated cattle (both 26 months). Significant changes in retinal function were observed in H-type BSE challenged cattle only. Cattle challenged with the same inoculum showed similar severity and neuroanatomical distribution of vacuolation and disease-associated prion protein deposition in the brain, though differences in neuropathology were observed between E211K BSE-H and classical BSE inoculated animals. Western blot results for brain tissue from challenged animals were consistent with the inoculum strains. This study demonstrates that the phenotype of E211K BSE-H remains stable when transmitted to cattle without the K211 polymorphism, and exhibits a number of features that differ from classical BSE in both wild-type and heterozygous EK211 animals.

A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection.

Infection with Bacillus anthracis, the causative agent of anthrax, can lead to persistence of lethal secreted toxins in the bloodstream, even after antibiotic treatment. VHH single-domain antibodies have been demonstrated to neutralize diverse bacterial toxins both in vitro and in vivo, with protein properties such as small size and high stability that make them attractive therapeutic candidates. Recently, we reported on VHHs with in vivo activity against the protective antigen component of the anthrax toxins. Here, we characterized a new set of 15 VHHs against the anthrax toxins that act by binding to the edema factor (EF) and/or lethal factor (LF) components. Six of these VHHs are cross-reactive against both EF and LF and recognize the N-terminal domain (LFN, EFN) of their target(s) with subnanomolar affinity. The cross-reactive VHHs block binding of EF/LF to the protective antigen C-terminal binding interface, preventing toxin entry into the cell. Another VHH appears to recognize the LF C-terminal domain and exhibits a kinetic effect on substrate cleavage by LF. A subset of the VHHs neutralized against EF and/or LF in murine macrophage assays, and the neutralizing VHHs that were tested improved survival of mice in a spore model of anthrax infection. Finally, a bispecific VNA (VHH-based neutralizing agent) consisting of two linked toxin-neutralizing VHHs, JMN-D10 and JMO-G1, was fully protective against lethal anthrax spore infection in mice as a single dose. This set of VHHs should facilitate development of new therapeutic VNAs and/or diagnostic agents for anthrax.

Stability properties of PrP(Sc) from cattle with experimental transmissible spongiform encephalopathies: use of a rapid whole homogenate, protease-free assay.

BACKGROUND: Transmissible Spongiform Encephalopathies (TSEs), including scrapie in sheep, chronic wasting disease (CWD) in cervids, transmissible mink encephalopathy (TME), and bovine spongiform encephalopathy (BSE), are fatal diseases of the nervous system associated with accumulation of misfolded prion protein (PrP(Sc)). Different strains of TSEs exist, associated with different PrP(Sc) conformations that can be probed by the stability assay, in which PrP(Sc) is treated with increasing concentrations of the denaturant guanidine hydrochloride (GdnHCl). RESULTS: Here, we provide the first comprehensive application of a rapid, protease-free version of the GdnHCl stability assay to brain tissue from cattle experimentally infected with various TSE isolates. Consistent with previous findings from a single Japanese isolate, the L-type isolates of BSE are not distinguishable from classical BSE in this assay. In contrast, H-type isolates of BSE, including our unique isolate of E211K BSE, exhibit higher stability than classical BSE, suggesting that its increased protection against protease digestion at the BSE N-terminus is associated with a higher stability in GdnHCl. While the difference in stability in our version of the assay is likely not large enough for effective use in a diagnostic laboratory setting, the use of alternative experimental conditions may enhance this effect. TSEs from other natural host species that have been passaged in cattle, including CWD and TME, were not distinguishable from classical BSE, while isolates of cattle passaged scrapie exhibited a slight increase in stability as compared to classical BSE. CONCLUSIONS: These results suggest that the core of PrP(Sc), as probed in this assay, has similar stability properties among cattle-passaged TSE isolates and that the conformational differences that lead to changes in the proteinase K cleavage site do not cause large changes in the stability of PrP(Sc) from TSE-affected cattle. However, the stability differences observed here will provide a basis of comparison for new isolates of atypical BSE observed in the future and in other geographic locations, especially in the case of H-type BSE.

The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and ß', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation.

Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiled-coil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.

Relationships between PrPSc stability and incubation time for United States scrapie isolates in a natural host system.

Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep (Ovis aries), are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP(C)) into a â-rich conformer (PrP(Sc)) that accumulates into higher-order structures in the brain and other tissues. Distinct strains of TSEs exist, characterized by different pathologic profiles upon passage into rodents and representing distinct conformations of PrP(Sc). One biochemical method of distinguishing strains is the stability of PrP(Sc) as determined by unfolding in guanidine hydrochloride (GdnHCl), which is tightly and positively correlated with the incubation time of disease upon passage into mice. Here, we utilize a rapid, protease-free version of the stability assay to characterize naturally occurring scrapie samples, including a fast-acting scrapie inoculum for which incubation time is highly dependent on the amino acid at codon 136 of the prion protein. We utilize the stability methodology to identify the presence of two distinct isolates in the inoculum, and compare isolate properties to those of a host-stabilized reference scrapie isolate (NADC 13-7) in order to assess the stability/incubation time correlation in a natural host system. We demonstrate the utility of the stability methodology in characterizing TSE isolates throughout serial passage in livestock, which is applicable to a range of natural host systems, including strains of bovine spongiform encephalopathy and chronic wasting disease.

A comparative analysis of rapid methods for purification and refolding of recombinant bovine prion protein.

Bacterially-produced recombinant prion protein (rPrP) is a frequently used model system for the study of the properties of wild-type and mutant prion proteins by biochemical and biophysical approaches. A range of approaches have been developed for the purification and refolding of untagged rPrP expressed as inclusion bodies in Escherichia coli, including refolding by dialysis and simultaneous on-column purification and refolding. In order to perform a higher-throughput analysis of different rPrP proteins, an approach that produces highly pure rPrP with a minimum of purification steps and a high yield per liter of induced bacterial culture is desired. Here, we directly compare purification approaches for untagged bovine rPrP as adapted to rapid, small-scale formats useful for higher-throughput studies. An analysis of protein yield, purity, oxidation, and refolding revealed significant differences between preparative methods as adapted to the small-scale format, and based on these findings we provide recommendations for future purifications. We also describe the utility of a sensitive commercial kit for thiol analysis of these preparations, the pH dependence of dimer formation during refolding of bovine rPrP, and bovine rPrP binding to cobalt affinity resin.

An improved procedure for the purification of the Escherichia coli RNA polymerase omega subunit.

We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only two chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and co-overexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.