Rnq1: an epigenetic modifier of protein function in yeast

Two protein-based genetic elements (prions) have been identified in yeast. It is not clear whether other prions exist, nor is it understood how one might find them. We established criteria for searching protein databases for prion candidates and found several. The first examined, Rnq1, exists in distinct, heritable physical states, soluble and insoluble. The insoluble state is dominant and transmitted between cells through the cytoplasm. When the prion-like region of Rnq1 was substituted for the prion domain of Sup35, the protein determinant of the prion [PSI+], the phenotypic and epigenetic behavior of [PSI+] was fully recapitulated. These findings identity Rnq1 as a prion, demonstrate that prion domains are modular and transferable, and establish a paradigm for identifying and characterizing novel prions.     

The Ubiquitin Proteasome System Plays a Role in Venezuelan Equine Encephalitis Virus Infection

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.     

Multigeneration Cross Contamination of Mail with Bacillus Species Spores by Tumbling

In 2001, envelopes loaded with Bacillus anthracis spores were mailed to Senators Daschle and Leahy as well as to the New York Post and NBC News buildings. Additional letters may have been mailed to other news agencies because there was confirmed anthrax infection of employees at these locations. These events heightened the awareness of the lack of understanding of the mechanism(s) by which objects contaminated with a biological agent might spread disease. This understanding is crucial for the estimation of the potential for exposure to ensure the appropriate response in the event of future attacks. In this study, equipment to simulate interactions between envelopes and procedures to analyze the spread of spores from a “payload” envelope (i.e., loaded internally with a powdered spore preparation) onto neighboring envelopes were developed. Another process to determine whether an aerosol could be generated by opening contaminated envelopes was developed. Subsequent generations of contaminated envelopes originating from a single payload envelope showed a consistent two-log decrease in the number of spores transferred from one generation to the next. Opening a tertiary contaminated envelope resulted in an aerosol containing 10³ B. anthracis spores. We developed a procedure for sampling contaminated letters by a nondestructive method aimed at providing information useful for consequence management while preserving the integrity of objects contaminated during the incident and preserving evidence for law enforcement agencies.In September and October of 2001, letters containing Bacillus anthracis spores were distributed through the U.S. Postal Service (USPS), resulting in contamination of the mail processing and distribution center in Hamilton, NJ, as well as affiliated processing centers in Washington, DC, in New York City, NY, and in Wallingford, CT, as well as postal facilities along the path transited by letters mailed to a targeted media company in Florida. Subsequently, 22 individuals, including postal workers, persons who received or handled the contaminated letters, and persons exposed to environments contaminated by the letters, developed cases of anthrax, including both the inhalation and cutaneous forms of the disease (5, 18-20). Five of these cases of anthrax resulted in death (4, 7). There have been investigations into the relationships of infection and exposure in areas where known exposures occurred (1, 6, 8). However, for two of the individuals who developed inhalational anthrax, an elderly woman in Connecticut and a nurse in New York City, no B. anthracis spores were detected (based on environmental sampling) on their mail or in their homes (2, 17, 19, 20). A third individual, a bookkeeper from New Jersey, survived a cutaneous anthrax infection, and only a single positive environmental sample in her workplace was identified (19).For the three specific cases mentioned above, the authors of the corresponding studies hypothesized that infection may have resulted from exposure to mail cross contaminated by mail that went through the same sorting equipment around the time that the letters to Senators Leahy and Daschle were processed. Without evidence of B. anthracis spores in their homes and other areas they were known to have frequented and the lack of additional cases in these geographic areas, there is no way to confirm the route of their exposure. We hypothesize that these people may have been exposed by inhaling spores released from envelopes that they tore open and then discarded. The delay between exposure and disease would have been sufficient to permit the discarded items to enter into the solid waste or recycling stream, and any residual spores may have been removed by normal housekeeping activities. Alternatively, the true source of exposure may have been undetectable due to a low concentration of spores.Those cases of anthrax raise the question of what, if any, hazards may have been encountered in handling mail with secondary and tertiary contamination. These cases raise particular questions concerning the ability of disease-causing organisms to spread through cross contamination of second- and even third-generation fomites in sufficient numbers to cause infection and possible death.Following the attacks, numerous studies were conducted in the contaminated postal buildings to assess the degree of contamination and to better understand sampling methodologies. Subsequent laboratory studies have been performed to improve B. anthracis sample collection and detection (11, 16, 22, 24, 30). Programs have monitored aerosols within federal buildings, hospitals, and mail facilities (10, 15, 25, 27). Additionally, studies of mail sorting machinery and the potential of this machinery to cross contaminate mail have been done (3, 10). However, to date, no laboratory studies that examined the potential for cross contamination of mail through contact or mixing with contaminated letters have been published.Reaerosolization in general is a poorly studied phenomenon. Characterization of reaerosolization under a variety of circumstances was undertaken following the B. anthracis incidents in 2001 (21, 29). The concept of fomite-to-fomite transference of powdered pathogen residues has been even less well studied.The settling of a primary aerosol comprised of charged particles may be due at least in part to an increase in the mass of these charged particles that occurs when they interact with oppositely charged particles. Once deposited on a surface, several factors may act against reaerosolization. Charged particles that have interacted with oppositely charged particles and have effectively increased in mass may be substantially more difficult to entrain in an aerosol than the initial particles. For charged particles that have not interacted with other particles, there may be a direct electrostatic interaction between the charged particle and the surface on which it has landed which would tend to hold these particles onto the surface. Both of these effects should reduce the potential for reaerosolization.Particulate preparations have a variety of properties, such as hydrophobicity, zeta potential, particle shape, and other characteristics that may also affect the potential for reaerosolization. It would be interesting to characterize a large number of powders, to create a database of the characteristics and their potential for aerosol formation and reaerosolization of these powders, and to use this database of information for comparison of unknown powders. Knowing this information may assist in the public health and risk management decision making processes. Unfortunately, there is no comprehensive database for these characteristics, nor is there any well-accepted unifying theory for deriving the likelihood of reaerosolization from the characteristics of powders that are commonly measured. In addition, there may be unknown variables that have an impact on aerosolization or reaerosolization that become known over time with improvements in understanding the theory of aerosolization and technology for measurement of these variables. A further confounding factor would be the inability to collect this information from the actual material used in any incident. In the case of the 2001 attacks (and likely in future incidents), there was (and will likely be) little material available for such study. The material used in the attacks is inherently hazardous and must be handled in highly controlled settings. The material is therefore difficult and expensive to work with (23). Material used in an attack is also generally sequestered as evidentiary material, and information concerning preparation of a biological weapon used in an attack may be considered too sensitive for public release. This sensitivity may include unwillingness to provide access to information on the efficacy of a specific preparation method to malevolent individuals and the requirement to preserve information for use in successful identification and prosecution of the perpetrator of such an attack. However, it may be possible to collect fomites contaminated with trace amounts of the agent in the course of public health investigations. The current study details a method for dealing with these contaminated fomites to yield information useful for public health protection.A confounding factor in these cases may be the necessity to treat as much of the available bulk material as can be collected as evidence. As evidence, even small amounts of this material may not be available for scientific testing. There may also be restrictions on the handling and treatment of fomites contaminated with residual traces of biological threat agents. For instance, the owners of the fomites may value them highly and may not wish to see them destroyed in the hope that the object may be somehow decontaminated and returned or the owner may wish to prevent public disclosure of the nature or contents of a contaminated object, such as a letter. It is therefore incumbent upon researchers to develop methods that are as minimally invasive and destructive as possible to investigate the potential for fomite-to-fomite transmission.We constructed a device designed to expose uncontaminated fomites to envelopes bearing a powdered preparation of spores or to fomites that had been exposed to other fomites contaminated by the initial powder-bearing envelope. Specifically, fomites used in this study were envelopes containing a piece of paper. This device was designed to conduct the exposure in a consistent, reproducible manner and to allow investigation of the interaction and cross contamination that might be encountered between a “payload” letter (a letter that had been loaded internally with a powdered spore preparation) and other pieces of mail. Uncontaminated envelopes were tumbled with a single envelope containing a payload of milled Bacillus atrophaeus subsp. globigii spores. After tumbling three successive generations of envelopes, CFU counts from the outsides of the envelopes were taken. These estimates of spore loads on the outside of these envelopes may be compared to published human 50% lethal dose (LD₅₀) estimates for aerosolized B. anthracis spores (12, 13). An additional series of envelopes was exposed to envelopes that had been contaminated during this first round of exposures, and those envelopes were found to be externally contaminated as well. We also studied opening an envelope that had been exposed to a payload envelope with either a finger or a letter opener to determine if these activities caused an aerosolization or reaerosolization of a sufficient number of spores to pose a risk of disease through inhalation.It is difficult to balance the concerns of making information public during a public health response and providing sufficient information for information risk management decision making while at the same time preserving the evidence for use by law enforcement agencies for eventual prosecution of individuals accused of committing crimes. We identified a nondestructive procedure by which contaminated mail can be analyzed and biological material collected while still preserving evidence for law enforcement agencies, allowing the payload envelope to be used as evidence while still permitting an assessment of its biological contaminant burden.     

Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis

Heat shock factor 1 (HSF1) is the master regulator of the heat shock response in eukaryotes, a very highly conserved protective mechanism. HSF1 function increases survival under a great many pathophysiological conditions. How it might be involved in malignancy remains largely unexplored. We report that eliminating HSF1 protects mice from tumors induced by mutations of the RAS oncogene or a hot spot mutation in the tumor suppressor p53. In cell culture, HSF1 supports malignant transformation by orchestrating a network of core cellular functions including proliferation, survival, protein synthesis, and glucose metabolism. The striking effects of HSF1 on oncogenic transformation are not limited to mouse systems or tumor initiation; human cancer lines of diverse origins show much greater dependence on HSF1 function to maintain proliferation and survival than their nontransformed counterparts. While it enhances organismal survival and longevity under most circumstances, HSF1 has the opposite effect in supporting the lethal phenomenon of cancer.     

Vertical transmission of a phylogenetically complex microbial consortium in the viviparous sponge Ircinia felix

Many marine demosponges contain large amounts of phylogenetically complex yet highly sponge-specific microbial consortia within the mesohyl matrix, but little is known about how these microorganisms are acquired by their hosts. Settlement experiments were performed with the viviparous Caribbean demosponge Ircinia felix to investigate the role of larvae in the vertical transmission of the sponge-associated microbial community. Inspections by electron microscopy revealed large amounts of morphologically diverse microorganisms in the center of I. felix larvae, while the outer rim appeared to be devoid of microorganisms. In juveniles, microorganisms were found between densely packed sponge cells. Denaturing gradient gel electrophoresis (DGGE) was performed to compare the bacterial community profiles of adults, larvae, and juvenile sponges. Adults and larvae were highly similar in DGGE band numbers and banding patterns. Larvae released by the same adult individual contained highly similar DGGE banding patterns, whereas larvae released by different adult individuals showed slightly different DGGE banding patterns. Over 200 bands were excised, sequenced, and phylogenetically analyzed. The bacterial diversity of adult I. felix and its larvae was comparably high, while juveniles showed reduced diversity. In total, 13 vertically transmitted sequence clusters, hereafter termed "IF clusters," that contained sequences from both the adult sponge and offspring (larvae and/or juveniles) were found. The IF clusters belonged to at least four different eubacterial phyla and one possibly novel eubacterial lineage. In summary, it could be shown that in I. felix, vertical transmission of microorganisms through the larvae is an important mechanism for the establishment of the sponge-microbe association.     

Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces

AIM: To evaluate US Centers for Disease Control and Prevention recommended swab surface sample collection method for recovery efficiency and limit of detection for powdered Bacillus spores from nonporous surfaces. METHODS AND RESULTS: Stainless steel and painted wallboard surface coupons were seeded with dry aerosolized Bacillus atrophaeus spores and surface concentrations determined. The observed mean rayon swab recovery efficiency from stainless steel was 0.41 with a standard deviation (SD) of +/-0.17 and for painted wallboard was 0.41 with an SD of +/-0.23. Evaluation of a sonication extraction method for the rayon swabs produced a mean extraction efficiency of 0.76 with an SD of +/-0.12. Swab recovery quantitative limits of detection were estimated at 25 colony forming units (CFU) per sample area for both stainless steel and painted wallboard. CONCLUSIONS: The swab sample collection method may be appropriate for small area sampling (10 -25 cm2) with a high agent concentration, but has limited value for large surface areas with a low agent concentration. The results of this study provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequence of an inappropriate public health response founded on information gathered using an ineffective or unreliable sample collection method has the potential for undesired social and economic impact.