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In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis Using RNA sequencing, we found that 2'3'-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis Many of the antiviral genes induced by 2'3'-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2'3'-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2'3'-cGAMP signaling in animals.

Significance Cyclic dinucleotides are signaling molecules that originated in bacteria and were subsequently acquired and co-opted by animals for immune signaling. The major cyclic dinucleotide signaling pathway in mammals results in the production of antiviral molecules called interferons. Invertebrates such as sea anemones lack interferons, and thus it was unclear whether cyclic dinucleotide signaling would play a role in immunity in these animals. Here, we report that in the anemone Nematostella vectensis, cyclic dinucleotides activate both antiviral and antibacterial immune responses and do so through a conserved pathway. These results provide insights into the evolutionary origins of innate immunity and suggest a broader ancestral role for cyclic dinucleotide signaling that evolved toward more specialized antiviral functions in mammals., In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2′3′ cyclic guanosine monophosphate-adenosine monophosphate (2′3′-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis. Using RNA sequencing, we found that 2′3′-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis. Many of the antiviral genes induced by 2′3′-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2′3′-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2′3′-cGAMP signaling in animals.

The ITER fusion reactor is being built to demonstrate the feasibility of fusion power and will be the largest tokamak in the world. The tokamak cooling water system (TCWS) will extract the ...

Heat generated in the ITER fusion reactor is deposited in the tokamak vacuum vessel, in-vessel components, and in the components of the neutral beam injector during plasma operations and du...

In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an anti-viral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2’3’-cGAMP, to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis. Using RNA-Seq, we found that 2’3’-cGAMP induces robust transcription of both anti-viral and anti-bacterial genes in N. vectensis. Many of the anti-viral genes induced by 2’3’-cGAMP are homologs of vertebrate interferon stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing anti-bacterial genes downstream of 2’3’-cGAMP. Some of these putative anti-bacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative anti-bacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved anti-bacterial activity. This work suggests that a broad anti-bacterial and anti-viral transcriptional response is an evolutionarily ancestral output of 2’3’-cGAMP signaling in animals.Significance statementCyclic dinucleotides are signaling molecules that originated in bacteria and were subsequently acquired and co-opted by animals for immune signaling. The major cyclic dinucleotide signaling pathway in mammals results in the production of anti-viral molecules called interferons. Invertebrates such as sea anemones lack interferons, and thus it was unclear whether cyclic dinucleotide signaling would play a role in immunity in these animals. Here we report that in the anemone Nematostella vectensis, cyclic dinucleotides activate both anti-viral and anti-bacterial immune responses, and do so through a conserved pathway. These results provide insights into the evolutionary origins of innate immunity, and suggest a broader ancestral role for cyclic dinucleotide signaling that evolved toward more specialized anti-viral functions in mammals.

The Oak Ridge National Laboratory High Flux Isotope Reactor (HFIR) is an 85 MWt flux trap-type research reactor that supports key research missions, including isotope production, materials irradiation, and neutron scattering. The core consists of an inner and an outer fuel element containing 171 and 369 involute-shaped plates, respectively. The thin fuel plates consist of a U3O8-Al dispersion fuel (highly enriched), an aluminum-based filler, and aluminum cladding. The fuel meat thickness is varied across the width of the involute plate to reduce thermal flux peaks at the radial edges of the fuel elements. Some deviation from the designed fuel meat shaping is allowed during manufacturing. A homogeneity scan of each fuel plate checks for potential anomalies in the fuel distribution by scanning the surface of the plate and comparing the attenuation of the beam to calibration standards. While typical HFIR simulations use homogenized fuel regions, explicit models of the plates were developed under the Low-Enriched Uranium Conversion Program. These explicit models typically include one inner and one outer fuel plate with nominal fuel distributions, and then the plates are duplicated to fill the space of the corresponding fuel element. Therefore, data extracted from these simulations are limited to azimuthally averaged quantities. To determine the reactivity and physics impacts of an as-built outer fuel element and generate azimuthally dependent data in the element, 369 unique fuel plate models were generated and positioned. This model generates the three-dimensional (i.e., radial–axial–azimuthal) plate power profile, where the azimuthal profile is impacted by features within the adjacent control element region and beryllium reflector. For an as-built model of the outer fuel element, plate-specific homogeneity data, 235U loading, enrichment, and channel thickness measurements were translated into the model, yielding a much more varied azimuthal power profile encompassed by uncertainty factors in analyses. These models were run with the ORNL-TN and Shift Monte Carlo tools, and they contained upwards of 500,000 cells and 100,000 unique tallies.

Neutronics analyses of the ITER experimental fusion reactor rely on increasingly complex geometry models and estimates of energy-dependent neutron flux and radiation dose-rate distributions generated at ever higher resolutions. There are significant practical challenges with applying the Monte Carlo N-Particle (MCNP) continuous-energy transport code to high-resolution analyses. For models consisting of more than 100 000 surfaces and cells, geometry initialization can take several hours, thus slowing down model integration and transport analysis efforts. In multithreaded simulations, the amount of memory consumed by superimposed mesh tally data increases in proportion to the number of threads. This behavior limits either the tally resolution or the number of processor cores that can be utilized in the simulation. This paper describes algorithmic improvements that were implemented in a modified version of MCNP5 to overcome these limitations. These improvements are referred to as the Oak Ridge Nation...

Fusion energy systems present increasingly significant computational challenges as they grow in size and complexity. Once constructed, ITER will be a full-size nuclear facility with highly complica...

The ITER International Organization has developed a number of reference Monte Carlo N-Particle (MCNP) models including the tokamak machine C-model, the Tokamak Complex model, and the neutral beam injection (NBI) systems model. The Tokamak Complex model primarily describes building structures beyond the bioshield. Representation of the tokamak and its systems are not included in this model. The Oak Ridge National Laboratory Radiation Transport Group has conducted two ITER neutronic analysis model integrations: (1) integration of the tokamak C-model with the Tokamak Complex model for shutdown dose rate characterization in Port Cell 16 at level B1, and (2) integration of the NBI model with the Tokamak Complex model for estimating the spatial distribution of biological dose rate at levels L1, L2, and L3 of the Tokamak Complex. The integrated models were further extended to include models of system components that are essential to the neutronic analyses. This paper presents the approach and computer to...

Detailed spatial distributions of the biological dose rate due to a variety of sources are required for the design of the ITER tokamak facility to ensure that all radiological zoning limits...

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA derived from bacteria, viruses and tumors. To signal, the C-terminal tail (CTT) of STING recruits TBK1, a kinase that phosphorylates serine 365 (S365) in the CTT. Phospho-S365 acts as a docking site for IRF3, a transcription factor that is phosphorylated and activated by TBK1, leading to transcriptional induction of type I interferons (IFNs). IFNs are essential for antiviral immunity and are widely viewed as the primary output of STING signaling in mammals. However, other more evolutionarily ancestral responses, such as induction of NF-κB or autophagy, also occur downstream of STING. The relative importance of the various outputs of STING signaling during in vivo infections is unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) point mutation in STING exhibit normal susceptibility to Mycobacterium tuberculosis infection but, unexpectedly, are resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. Likewise, we find Irf3-/- mice exhibit resistance to HSV-1. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT or TBK1, suggesting that STING protects against HSV-1 upon TBK1 recruitment by the STING CTT, independent of IRF3 or type I IFNs. Interestingly, we find that STING-induced autophagy is a TBK1-dependent IRF3-independent process that is conserved in the STING S365A mice, and autophagy has previously been shown to be required for resistance to HSV-1. We thus propose that autophagy and perhaps other ancestral interferon-independent functions of STING are required for STING-dependent antiviral responses in vivo.

SummaryIn humans, the cGAS-STING immunity pathway signals in response to cytosolic DNA via 2′,3′ cGAMP, a cyclic dinucleotide (CDN) second messenger containing mixed 2′–5′ and 3′–5′ phosphodiester bonds. Prokaryotes also produce CDNs, but these are exclusively 3′ linked, and thus the evolutionary origins of human 2′,3′ cGAMP signaling are unknown. Here we illuminate the ancient origins human cGAMP signaling by discovery of a functional cGAS-STING pathway in Nematostella vectensis, an anemone species >500 million years diverged from humans. Anemone cGAS appears to produce a 3′,3′ CDN that anemone STING recognizes through nucleobase-specific contacts not observed in human STING. Nevertheless, anemone STING binds mixed-linkage 2′,3′ cGAMP indistinguishably from human STING, trapping a unique structural conformation not induced by 3′,3′ CDNs. These results reveal that human mixed-linkage cGAMP achieves universal signaling by exploiting a deeply conserved STING conformational intermediate, providing critical insight for therapeutic targeting of the STING pathway.Graphical abstract

Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems.

Detection of foreign nucleic acids is an important strategy for innate immune recognition of pathogens. In vertebrates, pathogen-derived DNA is sensed in the cytosol by cGAS, which produces the cyclic dinucleotide (CDN) second messenger cGAMP to activate the signaling adaptor STING. While induction of antiviral type I interferons (IFNs) is the major outcome of STING activation in vertebrates, it has recently become clear that core components of the cGAS-STING pathway evolved more than 600 million years ago, predating the evolution of type I IFNs. Here we discuss the evolutionary origins of the cGAS-STING pathway, and consider the possibility that the ancestral functions of STING may have included activation of antibacterial immunity.

SummaryCyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2ʹ–5ʹ phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2ʹ–5ʹ cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2ʹ and 3ʹ linkage specificity and test this model by reprogramming the human cGAS active site to produce 3ʹ–5ʹ cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.

Cyclic di-nucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed fluorescent RNA-based biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic di-nucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.

© 2016 The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. Bacteria occupy a diverse set of environmental niches with differing oxygen availability. Anaerobic environments such as mammalian digestive tracts and industrial reactors harbor an abundance of both obligate and facultative anaerobes, many of which play significant roles in human health and biomanufacturing. Studying bacterial function under partial or fully anaerobic conditions, however, is challenging given the paucity of suitable live-cell imaging tools. Here, we introduce a series of RNA-based fluorescent biosensors that respond selectively to cyclic di-GMP, an intracellular bacterial second messenger that controls cellular motility and biofilm formation. We demonstrate the utility of these biosensors in vivo under both aerobic and anaerobic conditions, and we show that biosensor expression does not interfere with the native motility phenotype. Together, our results attest to the effectiveness and versatility of RNA-based fluorescent biosensors, priming further development and application of these and other analogous sensors to study host-microbial and microbial-microbial interactions through small molecule signals.

Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3′-5′, 3′-5′ cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

An environmental microbiologic investigation was conducted in an alligator (Alligator mississippiensis) holding facility in a zoo in the southeastern U.S. The facility had housed five alligators between March 1999 and February 2005. In the exhibit, one alligator died and all experienced poor health. It was hypothesized that environmental microbial contamination was associated with these issues. Samples were collected for fungal identification and quantification, microcystin analysis, and airborne mycotoxins. Analyses of air and water were conducted and an examination of the heating, ventilation, and air-conditioning system (HVAC) for design, maintenance, and operating issues was made. Two control sites, a facility for false gharials (Tomistoma schlegelii) and an off-site alligator breeding facility, were also tested. Morbidity and mortality records were examined for all sites. Results showed that, compared to the control sites, the test alligator facility and its HVAC system were extensively contaminated with a range of fungi. Nearly all sampled surfaces featured fungal growth. There were also significantly higher counts of Penicillium/Aspergillus-like and Chrysosporium-like spores in the air (P < 0.004). The design, maintenance, and operation of the HVAC system were all inadequate, resulting in poorly conditioned and mold-contaminated air being introduced to the facility. Morbidity records revealed solitary pulmonary disorders over time in three alligators, with one dying as a result. The other two alligators suffered from general malaise and a range of nonspecific symptoms. The control facilities had no morbidity or mortality issues. In conclusion, although no causal links could be demonstrated because of the nature of the morbidity data, environmental mold contamination appeared to be associated with the history of morbidity and mortality in the alligator exhibit.

The existence of airborne mycotoxins in mold-contaminated buildings has long been hypothesized to be a potential occupant health risk. However, little work has been done to demonstrate the presence of these compounds in such environments. The presence of airborne macrocyclic trichothecene mycotoxins in indoor environments with knownStachybotrys chartarumcontamination was therefore investigated. In seven buildings, air was collected using a high-volume liquid impaction bioaerosol sampler (SpinCon PAS 450-10) under static or disturbed conditions. An additional building was sampled using an Andersen GPS-1 PUF sampler modified to separate and collect particulates smaller than conidia. Four control buildings (i.e., no detectableS. chartarumgrowth or history of water damage) and outdoor air were also tested. Samples were analyzed using a macrocyclic trichothecene-specific enzyme-linked immunosorbent assay (ELISA). ELISA specificity was tested using phosphate-buffered saline extracts of the fungal generaAspergillus,Chaetomium,Cladosporium,Fusarium,Memnoniella,Penicillium,Rhizopus, andTrichoderma, fiveStachybotrysstrains, and the indoor air allergens Can f 1, Der p 1, and Fel d 1. For test buildings, the results showed that detectable toxin concentrations increased with the sampling time and short periods of air disturbance. Trichothecene values ranged from 1,300 pg/m3of sampled air. The control environments demonstrated statistically significantly (P< 0.001) lower levels of airborne trichothecenes. ELISA specificity experiments demonstrated a high specificity for the trichothecene-producing strain ofS. chartarum. Our data indicate that airborne macrocyclic trichothecenes can exist inStachybotrys-contaminated buildings, and this should be taken into consideration in future indoor air quality investigations.

The efficacy of chlorine dioxide (ClO2) in detoxifying two potential bioterrorism agents, the trichothecene mycotoxins verrucarin A and roridin A, was evaluated. In the first experiment, verrucarin A (1, 5, or 10 μg) and roridin A (5 or 10 μg) were each inoculated onto square-inch sections of glass, paper, and cloth and exposed to 1000 ppm of ClO2 for either 24 or 72 h at room temperature. In the second experiment, verrucarin A and roridin A (1 or 2 ppm in water) were treated with 200, 500, or 1000 ppm ClO2 for up to 116 h at room temperature in light and dark conditions ( N = 9 per treatment for test and control). A yeast assay using Kluyveromyces marxianus was used to quantify the toxicity of verrucarin A and roridin A. Additionally, high-performance liquid chromatography was performed on selected samples. Results for the first experiment showed that ClO2 treatment had no detectable effect on either toxin. For the second experiment, both toxins were completely inactivated at all tested concentrations in as little as 2 h after treatment with 1000 ppm ClO2. For verrucarin A, an effect was seen at the 500 ppm level, but this effect was not as strong as that observed at the 1000 ppm level. Roridin A toxicity was decreased after treatment with 200 and 500 ppm ClO2, but this was not significant until the 24-h exposure time was reached. These data show that ClO2 (in solution) can be effective for detoxification of roridin A or verrucarin A at selected concentrations and exposure times.

Highly respirable particles (diameter

Two experiments were conducted regarding the culturability and toxicity of fungi located on building materials over time and the efficacy of seven laboratory techniques in recovering culturable fungi from sample swabs. In the first experiment, eight sections of drywall were inoculated with Stachybotrys chartarum and stored at 25 +/- 5 degrees Celsius and 20-60% relative humidity (RH) for up to two years. Another eight sections of ceiling tile were stored at 100% RH for 1 year. Six sections of ceiling tile and 15 swabs were also inoculated with Penicillium chrysogenum and S. chartarum respectively and stored under the same conditions for 8 months and 3.3 years. All materials were tested for culturability at the end of the storage period. S. chartarum-inoculated samples were also tested for toxicity. In the second experiment (replicated twice), S. chartarum and Chaetomium globosum were inoculated onto 84 swabs each. Storage was up to 266 days at 25 +/- 5 degrees Celsius and 20-60% RH. Seven techniques were compared regarding the recovery of culturable fungi from the swabs over different time points. Results for Experiment 1 showed that all samples were culturable after the storage period and that the S. chartarum-inoculated drywall samples were toxic. In Experiment 2, all techniques showed high rates of recovery. These data show that despite being without a water source, these organisms can be culturable and toxic after long periods of time under conditions similar to human-occupied dwellings and that a number of preparation techniques are suitable for the recovery of these fungi from inoculated swabs.

We report on the first direct investigation of the low-energy electron-induced production of neutral species from the chlorofluorocarbon CF 2 Cl 2 , commonly known as Freon-12 or CFC-12. Our experiments were motivated by a newly proposed hypothesis, which suggests that low-energy electrons produced by cosmic rays, in addition to UV-vis photons from the sun, interact with chlorofluorocarbons to produce chlorine atoms that subsequently destroy ozone in the Antarctic. Our experimental procedure involves low-energy (5-100 eV) electron irradiation of nanoscale thin films (∼10 A thickness) of CF 2 Cl 2 grown at 100 K on a molybdenum single crystal in an ultrahigh vacuum chamber (p ∼ 1 × 10 - 1 0 Torr). Post-irradiation temperature-programmed desorption experiments were used to identify C 2 F 4 Cl 2 , C 2 F 3 Cl 3 , C 2 F 2 Cl 4 , C 2 F 3 Cl, C 2 F 2 Cl 2 , and C 2 F 4 as electron-induced radiolysis products of CF 2 Cl 2 . In contrast to previous studies of photon-induced dissociation, our studies of electron-induced dissociation demonstrate facile C-F bond cleavage in CF 2 Cl 2 . This finding may have implications for understanding the partitioning of Cl and F among source, sink, and reservoir gases in the stratosphere.

A total of 110 sites from five zoological institutions were examined to determine whether fungi associated with sick building syndrome (SBS) were prevalent in the exhibits or night-time holding facilities and to investigate whether the presence of these organisms was associated with declining breeding rates or increases in morbidity and mortality (or both). Each site was sampled with an Andersen two-stage air sampler using Sabourauds dextrose agar media and a Burkard personal volumetric air sampler. Suspect surfaces were also sampled. High levels of airborne Penicillium chrysogenum, a fungal species associated with poor indoor air quality, were recovered from 16 sites out of all five institutions. Five culturable growth sites of Stachybotrys chartarum, a species strongly associated with SBS and commonly known as "black mold," were recovered from surfaces at two institutions. A wide range of other fungal species was recovered in low numbers from all institutions. A Fisher exact test analysis showed a significant nonrandom association between high levels of P. chrysogenum and sites with records of poor animal health. This study indicated that significant numbers of airborne fungi associated with SBS and poor indoor air quality are present in zoological institutions and that they could affect animal health and reproduction rates and zoo staff.

Riboswitch aptamers adopt diverse and complex tertiary structural folds that contain both single-stranded and double-stranded regions. We observe that this high degree of secondary structure leads to an appreciable hypochromicity that is not accounted for in the standard method to calculate extinction coefficients using nearest-neighbor effects, which results in a systematic underestimation of RNA concentrations. Here we present a practical method for quantifying riboswitch RNAs using thermal hydrolysis to generate the corresponding pool of mononucleotides, for which precise extinction coefficients have been measured. Thermal hydrolysis can be performed at neutral pH without reaction quenching, avoids the use of nucleases or expensive fluorescent dyes, and does not require generation of calibration curves. The accuracy of this method for determining RNA concentrations has been validated using quantitative 31P-NMR calibrated to an external standard. We expect that this simple procedure will be generally useful for the accurate quantification of any sequence-defined RNA sample, which is often a critical parameter for in vitro binding and kinetic assays.

Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high-throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for benchtop assays (3.2 versus 1020 min), we screen and validate five candidate SAM-I riboswitches isolated from thermophilic and cryophilic bacteria. The format offers enhanced resolution of conformational change compared to slab gel formats, quantitation, and repeatability for statistical assessment of small mobility shifts, low reagent consumption, and riboswitch characterization without modification of the aptamer structure. Appreciable analytical sensitivity coupled with high-resolution separation performance allows quantitation of equilibrium dissociation constants (K(d)) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements, and K(d) are reported for both a known and a candidate SAM-I riboswitch with comparison to in-line probing assay results. The microfluidic mobility shift assay establishes a scalable format for the study of riboswitch-ligand binding that will advance the discovery and selection of novel riboswitches and the development of antibiotics to target bacterial riboswitches.

Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 13 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 13 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.

An investigation was conducted on selected locations in air handling units (AHUs) to (a) identify common mold species found on these locations, (b) determine whether some locations (and subsets) featured mold growth sites more frequently than others, (c) ascertain whether the operating condition of AHUs is related to mold contamination, and (d) provide a basis for a microbial sampling protocol for AHUs. A total of 566 tape lifts and 570 swab samples were collected from the blower wheel fan blades, insulation, cooling coil fins, and ductwork from 25 AHUs. All AHU conditions were numerically rated using a heating, ventilation and air-conditioning (HVAC) survey. Results showed that Cladosporium sp. fungi were commonly recovered in terms of growth sites and deposited spores, and they were found mainly in the blower wheel fan blades, the ductwork, and the cooling coil fins. Subsections of the fan blades, insulation, and cooling coil fins showed no preferred area for mold growth sites. Other organisms such as Penicillium sp., Aspergillus sp., and Paecilomyces sp. were recovered from the cooling coil fins and insulation. Because of the widespread prevalence of Cladosporium sp., there was no relationship between mold growth and operating condition. However, the presence of different species of molds in locations other than the blower wheel blades may indicate that the AHU condition is not optimal. A suggested microbial sampling protocol including interpretations of sample results is presented.

To date, no study has effectively demonstrated a direct human exposure to mycotoxins in mold-contaminated buildings. Therefore, the authors investigated the presence of trichothecene mycotoxins in sera from individuals exposed to indoor molds (specifically Stachybotrys chartarum). Sera from occupants of contaminated (test samples, n=44) and uncontaminated (control samples, n=26) buildings were analyzed using a competitive enzyme-linked immunosorbent assay (ELISA) highly specific for macrocyclic trichothecenes. Twenty-three samples were significantly different (p < 0.05) from normal human serum tested in the same manner, whereas only 1 of the control samples tested positive. Mass spectrometry analysis could not confirm the presence of intact S. chartarum macrocyclic trichothecenes. The authors hypothesize that this result was caused by uncharacterized ELISA-reactive metabolic breakdown products. Data from this study suggest that trichothecene mycotoxins can be demonstrated in the tissues of certain individuals exposed to S. chartarum in contaminated buildings.

This study used spectral analysis in 2 separate experiments to examine feeding and lying behavior of Bos taurus steers under 2 housing treatments: a feedlot yard stocked at 12.0 m2 per head with a dry, firm pen surface (NDF) and a "high density" (HDF) feedlot yard stocked at 6.0 m2 per head with a wet and muddy feedlot pen surface. The study conducted 1 experiment in Autumn, another in Spring. The study measured and analyzed ambient temperatures, relative humidity, and barometric pressure half-hourly onsite, using time series cross-correlations to determine whether steer behavior was associated with them. Both NDF and HDF groups showed similar lying and eating duration. HDF steers exhibited patterns of lying and feeding different from those of NDF steers. Spring observations found a number of correlations with temperature, relative humidity, and barometric pressure. Health and production data showed no differences between treatments. The results indicate that cattle made successful, short-term changes to changed feedlot environmental conditions. However, confirming these findings requires further replication. Spectral analysis was shown to be sensitive enough to detect behavioral differences between treatments and thus has potential animal welfare assessment tool.

Objective—To determine the bacterial, fungal, and endotoxin concentrations in aerosolized ambient air during the winter and summer in feedyards located in the Southern High Plains, identify aerosolized microbial pathogens, and determine the size of microbial and dust components. Sample Population—Aerosol samples were obtained from 7 feedyards. Procedure—Aerosol samples were collected upwind, on-site, and downwind from each feedyard at a point 1 m above the ground by use of biological 2- and 6- stage cascade impactors. Results—Significantly more microbes were cultured from on-site and downwind samples than upwind samples. There were significantly more microbes during the summer than during the winter. However, mean endotoxin concentration was significantly higher during the winter (8.37 ng/m3) than the summer (2.63 ng/m3). Among 7 feedyards, mean ± SE number of mesophilic bacteria (1,441 ± 195 colony-forming units [CFUs]/m3) was significantly higher than mean number of anaerobic bacteria (751 ± 133 CFUs/m3) or thermophilic bacteria (54 ± 10 CFUs/m3) in feedyard air. Feedyard aerosol samples contained more mesophilic fungi (78 ± 7 CFUs/m3) than thermophilic fungi (2 ± 0.2 CFUs/m3). Eighteen genera of bacteria were identified by use of an automated identification system. Conclusions and Clinical Relevance—It appeared that gram-negative enteric pathogens offered little risk to remote calves or humans via ambient aerosols and that gram-positive pathogens of the Bacillus, Corynebacterium, and Staphylococcus spp can be spread by aerosols in and around feedyards. It was common to detect concentrations of endotoxin in the ambient air of 7 feedyards. ( Am J Vet Res 2004; 65:45–52)

Publisher Summary This chapter presents a case study in which a series of protocols was used to identify mold-related problems in a school building complex. Techniques such as dehumidification of the outside air for the heating, ventilation, air conditioning (HVAC) system, the reconditioning of the HVAC system, and the physical removal of mold growth sites were used in the remediation process. Post-remediation monitoring processes indicate that these techniques have been successful. The HVAC system is the primary means for maintaining control of moisture in a dry building. It is also the primary transport mechanism for airborne pollutants. Once the conditions are suitable for fungal growth to be established in a building, the HVAC system can transport pollutants to other potential growth areas. In the case study, the HVAC system is examined for design flaws, construction defects, and histories of operational failures.

Publisher Summary Damp and moldy structures are potentially harmful to the occupants. A related major and largely uninvestigated subject also relevant to the occupants of mold-contaminated structures is the status of their contents inside these structures. The possibility exists that the contents may be contaminated by fungal colonies or conidia and mycotoxins. This chapter discusses the issue of the contents potentially being contaminated by mold conidia and mycotoxins, presents some recent research performed in this area, and examines some sterilizing procedures that have been found to be effective in eliminating fungal conidia and mycotoxins on home contents. The obstacles regarding the determination of levels of conidia/mycotoxins and fungal fragments on contents are such that it is possible that those levels are never determined. The strategy in light of this deficiency is to assume that the contents can potentially have high numbers of conidia, mycotoxins, or fragments and to use a cleaning technique on them. Although no technique is perfect, a few can inactivate conidia and mycotoxins from a wide range of organisms.

In a lag analysis, the frequencies of various events that follow a target or criterion event are tallied according to the ordinal position of each event observed following the target event. For behavior analysts, lag analysis is most useful for detecting regular patterns of contingent events, since reinforcers and punishers can be both identified and evaluated by their effects on antecedent and consequent behaviors. Once contingent events have been identified it is of considerable interest to detect extended sequences of particular events or classes of events. This article describes two FORTRAN programs that can facilitate the analysis of observational data: One, JOINT, computes the simple frequencies of events and provides a lag-sequential analysis; the other, SEQU, detects predefined sequences of events. Sackett, Holm, Crowley, and Henkins (1979) have recently described a FORTRAN program, LAGS, that performs lag-sequential analysis on behavioral interaction data, and they have addressed a number of theoretical issues involved in coding observational data and in applying inferential statistics to those data. JOINT shares many common features with LAGS and is offered here only as an alternative. There are two major differences between the two programs, nonetheless. First, in JOINT, all behaviors serve as the target behaviors in the lag analysis, whereas in LAGS only one target can be specified at a time. Second, in JOINT, the maximum lag allowed is 10, whereas LAGS can handle lags of up to 800. These two differences seem to result because JOINT passes through the data only once, but LAGS passes through the data separately for each target behavior. Thus, one program requires no a priori decisions about target behaviors, and the other allows a more extended lag analysis. Program Input. The input to both JOINT and SEQU is in two parts, or meso The first me, a control file, provides a general title, the numerical codes that represent the behaviors and alphanumeric labels for each, the lags to be analyzed, and an optional format statement for reading the second me, the data file. If any of the numerical codes represent more than one behavior that is coded in the data, the recoding information is given here. The second me, the data file, begins with a line of identifying information for the session and, in sequence, the numerical codes for each behavior recorded during the session. A unique code marks the end of the session, and another code marks the end of the set of sessions to be analyzed. JOINT accepts observational data that consist of up to 80 different codes, each code being a number that represents a particular behavior. At the user's option, codes may be assigned that represent the occurrence of any I of up to 80 other uniquely coded behaviors. Thus, the raw data may comprise many codes, but the program can analyze only 80 codes at a time. Memory limitations on some computers may require program modifications that will reduce the number of codes per analysis to 20 or 40. The data to be analyzed may be drawn from a single session or may be combined across as many as 50 different sessions. The number of behaviors recorded in a session is limited by only the largest integer value the computer can store, a value usually so large as to pose no practical problems. Program Output: JOINT. JOINT computes simple frequencies and probabilities and rates for each of the codes defined in the control file. JOINT also computes joint frequencies, conditional probabilities, z scores, and rates for the codes with lags from I to 10. The lag analysis is printed in matrix form, with the entire set of codes comprising both the row (R) and column (C) behaviors. For a lag of 1, the joint frequency of Rand C, f(R & C), is the number of times Behavior C occurred immediately after Behavior R occurred. For a lag of 2, of course, feR & C) is the number of times C was the second behavior recorded after R occurred, and so on. The conditional probability, p(C/R), is the probability of the C behavior, given that the R behavior was occurring in a preceding time frame. The conditional probability of a particular Rand C is obtained by dividing feR & C) by f(R). The rate of each behavior is calculated simply from the duration of the observation periods. Likewise, the rate of each joint frequency is given, that is, the rate at which a C behavior followed an R behavior at each lag. The z scores indicate the extent to which an observed joint frequency deviated from the joint frequency that would occur if Behavior Rand Behavior C were independent of each other. Assuming independence (or only chance association), the expected