Search

Airborne murine coronavirus was assessed for its sensitivity to the vapors of chemicals commonly used to disinfect indoor surfaces. As a model for the chemical sensitivity of airborne SARS-CoV-2, the infectious potential of airborne Mouse Hepatitis Virus (MHV) was tracked in the presence of the following pure chemical vapors, each of which was below its permissible exposure limit (PEL) as regulated by the US National Institute of Occupational Safety and Health (NIOSH): < 50 ppmv for glycol; < 1ppmv for HOCl; and, < 1ppmv for H2O2. Along with its growth media, infectious MHV was aerosolized in a particle size distribution between 0.5 μm and 3.2 μm into a sealed, dark, 9 m3 chamber maintained at 220C and 60% RH, including levels of chemical vapors maintained below their respective PELs. As judged by the TCID50 of airborne MHV collected by condensation, this airborne virus was rapidly inactivated by HOCl vapor, incurring an average of 99% infectious potential loss after 16 ± 4 minutes exposure to ≤0.2 ppmv HOCl. Airborne MHV responded with a 99% loss of infectious potential in 38 ± 10 minutes of exposure to ≤0.9 ppmv H2O2; and, a 99% loss of infectious potential in 33 ± 15 minutes when exposed to a gas-phase dipropylene glycol blend ≤20 ppmv as TVOC. The juxtaposition of quantitative RT-PCR and TCID50 responses suggest that even low levels of gas-phase HOCl exposures can damage the genome of airborne coronavirus in relatively short time frames (c.a. < 5 mins).

Many epidemiological studies have associated bioaerosol exposures with a variety of adverse health effects; however, the role of bioaerosol components in the development and manifestation of hypersensitivity and non-infectious respiratory diseases remains unclear. Despite many studies which have examined allergic responses to bioaerosols, less is known about non-allergenic effects. In order to elucidate the mechanisms by which bioaerosols can exert non-atopic stresses on a cellular level, there is a need for improving existing in vitro approaches. In response, a cohort of toxicology assays were optimized to create a robust analytical suite for studying the effects that biogenic atmospheric pollutants generate on two model human lung cell lines (A549 epithelial line and GDM-1 immature macrophage line). To demonstrate the utility for studying the cellular responses to select bioaerosols, cells exposed to curdlan (a linear (1 → 3)-β-glucan) were examined in a composite cytometry platform. Results suggest that curdlan has the potential to elicit significant responses in A549 and GDM-1 in two or more toxicological modes associated with exposure to airborne particulate matter. As designed, this suite provided a more powerful tool for characterizing curdlan-induced toxicological potential than any individual assay. Responses to curdlan were distinctly modal and cell line dependent, suggesting that the use of a suite of toxicological assays, in a common platform on different cell lines, can help provide important insights into the formative toxigenic responses that primary bioaerosols can induce in respiratory cells.

Rapid bioaerosol characterization has immediate applications in the military, environmental and public health sectors. Recent technological advances have facilitated single-particle detection of fluorescent aerosol in near real time; this leverages controlled ultraviolet exposures with single or multiple wavelengths, followed by the characterization of associated fluorescence. This type of ultraviolet induced fluorescence has been used to detect airborne microorganisms and their fragments in laboratory studies, and it has been extended to field studies that implicate bioaerosol to compose a substantial fraction of supermicron atmospheric particles. To enhance the information yield that new-generation fluorescence instruments can provide, we report the compilation of a referential aerobiological catalogue including more than 50 pure cultures of common airborne bacteria, fungi and pollens, recovered at water activity equilibrium in a mesoscale chamber (1 m3). This catalogue juxtaposes intrinsic optical properties and select bandwidths of fluorescence emissions, which manifest to clearly distinguish between major classes of airborne microbes and pollens.

In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C.

Small alterations in static magnetic fields have been shown to affect certain chemical reaction rates ex vivo. In this manuscript, we present data demonstrating that similar small changes in static magnetic fields between individual cell culture incubators results in significantly altered cell cycle rates for multiple cancer-derived cell lines. This change as assessed by cell number is not a result of apoptosis, necrosis, or cell cycle alterations. While the underlying mechanism is unclear, the implications for all cell culture experiments are clear; static magnetic field conditions within incubators must be considered and/or controlled just as one does for temperature, humidity, and carbon dioxide concentration.

The goal of this study was to design, test, and model cylindrical flow-through control devices used to inactivate airborne microorganisms with non-mercury radiation sources and a reflective coating, through a broad relative humidity (RH) range. Bench-scale tests determined the effectiveness of three different UV-C sources–light emitting diode (LED), xenon, and mercury. In addition to measurements, modeling was performed that combined photon tracing, computational fluid dynamics, and particle tracking to predict the effectiveness of the respective UV challenges. Regardless of device configuration, UV-induced inactivation of airborne Bacillus subtilis and Mycobacterium parafortuitum was markedly sensitive to low humidity (15 % RH). Actinometry measurements determined that a high reflectance wall coating increased the fluence rate by a factor of 1.6. Predictions of inactivation effectiveness using this hybrid model were in good agreement with experimental observations with errors of less than 15 %.

Interstrand crosslinks (ICLs) are a potent form of DNA damage in which the strands are covalently linked by a bifunctional chemical. The result is a block of normal DNA replication and transcription. The most readily available template for repair, the opposing strand, is also involved with the damage, complicating error-free repair. Straight forward excision and gap filling, as with nucleotide excision repair (NER), seems to be precluded. The many steps involved in IC makes ICL- inducing agents attractive as chemotheraputic drugs (e.g. cisplatin). However, not all ICLs are products of exogenous chemicals; ICLs can also be created by byproducts of metabolism, including the lipid peroxidation product malondialdehyde (Minko et al., 2008). Thus, we find ICL repair mechanisms in organisms from bacteria through humans. In E. coli, the ICL repair pathway utilizes both nucleotide excision repair (NER) and homologous recombination (HR) (Cole, 1973), apparently acting in a single pathway. UvrABC incises the DNA 5′- and 3′- of the ICL on one strand, and the 5′-exonuclease activity of DNA polymerase I creates a single stranded DNA (ssDNA) region required for RecA-mediated recombination. Strand invasion creates a structure on which UvrABC can act, removing the ICL-containing DNA fragment (Dronkert and Kanaar, 2001). The gap is filled by DNA polymerase I and covalently bonded by polynucleotide ligase. In Sacchromyces cerevisiae, studies have shown many genes are involved in ICL repair. Genetic evidence indicates there are three distinct ICL repair pathways in S. cerevisiae, representing ‘NER’, post-replication repair, and HR represented by SNM1, REV3, and RAD51 epistasis groups respectively (Grossmann et al., 2001). Apparently early in ICL repair double strand breaks (DSB) are formed as intermediates (Jachymczyk et al., 1981). Subsequently, a recombination-dependent step utilizing RAD51 may act in completing ICL repair, through repair of the DSB (Jachymczyk et al., 1981). This occurs only when a homologous sequence is available. Post-replication/translesion synthesis utilizing error-prone polymerases such as ζ (REV3/Rev7) or η might replicate past the ICL following DSB formation (Jachymczyk et al., 1981). While rev3 mutants are sensitive to ICL damage, yeast DNA polymerase η mutants show normal sensitivity to ICL, suggesting no role for this bypass polymerase in repair (Grossmann et al., 2001). Less is known about this pathway than NER and HR repair; however, it appears that the pathway allows cells to bypass an ICL as opposed to actually repairing the lesion (Dronkert and Kanaar, 2001). Yeast snm1 Δ mutants are specifically sensitive to ICL (Henriques et al., 1997) but display normal incision (Li and Moses, 2003). They do not, however, resolve the DSB and restore high molecular weight DNA after cross-links (Magana-Schwencke et al., 1982) (Li et al., 2005). Apparently the SNM1 protein, known to be a 5’-exonuclease (Li et al., 2005), acts to modify intermediates of DSB repair. Interestingly, snm1 mutants show normal processing of mating type, indicating that the DSBs occurring in that pathway are processed normally. This leads to the conclusion that the DSBs arising during the ICL repair process have a specific structure, different from DSBs created in mating type switching, thus requiring different processing. As noted, ICL repair appears to involve DSB intermediates; the processing of the DSB resulting from ICL repair requires specific activities peculiar to the process. Therefore, while RAD51, for example, is required for repair of DSBs after ionizing radiation or ICLs, SNM1 is required only for ICL DSB repair. Such a comparison illustrates that some components of ICL repair may act in a general DSB response whereas others act specifically. It appears that the DSBs occurring during normal DNA replication use many of the same components that ICL repair utilizes for genome stability and provide a substrate for HR (Ward et al., 2007). The mammalian ICL repair pathway is more complex than E. coli or yeast. Like yeast, ICL repair operates through a DSB intermediate similar to those occurring in S-phase during replication [reviewed in (Patel and Joenje, 2007)]. While NER and HR pathways are implicated in ICL repair (De Silva et al., 2000; Zheng et al., 2003), other proteins, not found in E. coli or S. cerevisiae are involved. The Fanconi anemia (FA) pathway, as demonstrated by the extreme sensitivity of patients, model organisms, and cell lines to ICLs, is involved in mammalian ICL repair [reviewed in (Kennedy and D’Andrea, 2005; Patel and Joenje, 2007; Wang, 2007)]. Activation of the FA pathway occurs during normal S-phase, likely in response to DSBs or other events during replication, and also occurs with hydroxyurea (HU) treatment which results in stalled replication forks with no ICLs, leading to DSBs (Diffley et al., 2000; Fox, 1985). These observations suggest that DSBs caused by HU (substrate depletion) may be equivalent to those arising from ICL repair. FA patients also exhibit chromosome instability, leading to chromosomal radial formations (Figure 1), levels of which are increased as a result of ICL exposure. In fact, radials are not observed in normal cells without ICL damage at high levels. This implies an overload of the repair system in FA cells. The radial formations may occur as ‘quadriradials’ showing symmetry of four arms, or as adhesions of less than two complete sets of chromatids. The finding of radials is accompanied by apparent breaks in chromatids (Figure 1). Figure 1 Metaphase spread demonstrating chromosome radials. The participation of multiple repair pathways in ICL repair raises the question of whether there is a single monolithic repair response to ICLs in mammals, or if the pathways are independent, as has been unambiguously shown for yeast (Grossmann et al., 2001; McHugh et al., 2001). While models for a unitary path involving NER, HR and post-replication have been suggested (Niedernhofer et al., 2005), results from mutant cell lines and siRNA depletion indicate there are multiple independent pathways in mammals (Hanlon Newell et al., 2008; Hemphill et al., 2008; Wang, 2007) (Jakobs, unpublished). In addition, it appears several bypass DNA polymerases, including Pol κ, may be involved (Minko et al., 2008), not just Pol ζ, further complicating interpretaion.

Epidemiological studies have shown that exposure to airborne particulate matter can be an important risk factor for some common respiratory diseases. While many studies have shown that particulate matter exposures are associated with inflammatory reactions, the role of specific cellular responses in the manifestation of primary hypersensitivities, and the progression of respiratory diseases remains unclear. In order to better understand mechanisms by which particulate matter can exert adverse health effects, more robust approaches to support in vitro studies are warranted. In response to this need, a group of accepted toxicology assays were adapted to create an analytical suite for screening and evaluating the effects of important, ubiquitous atmospheric pollutants on two model human lung cell lines (epithelial and immature macrophage). To demonstrate the utility of this suite, responses to intact diesel exhaust particles, and mass-based equivalent doses of their organic extracts were examined. Results suggest that extracts have the potential to induce greater biological responses than those associated with their colloidal counterpart. Additionally, macrophage cells appear to be more susceptible to the cytotoxic effects of both intact diesel exhaust particles and their organic extract, than epithelial cells tested in parallel. As designed, the suite provided a more robust basis for characterizing toxicity mechanisms than the analysis of any individual assay. Findings suggest that cellular responses to particulate matter are cell line dependent, and show that the collection and preparation of PM and/or their extracts have the potential to impact cellular responses relevant to screening fundamental elements of respiratory toxicity.

The diversity of applications utilizing antimicrobial laden textiles continues to grow, yet testing methods based on the liquid loading of cultures to challenge textiles remain unchanged. For bioaerosol applications, liquid challenge methods are unsuitable. We present a method of aerosol based loading and microbial recovery for contextual testing antimicrobial textiles.

Goblet cell carcinoid (GCC) is a rare but distinctive neoplasm with features of both adenocarcinoma and carcinoid tumor. Most cases described in the literature have occurred in the appendix. An additional well-defined location is the ovary, and these tumors have been associated with a mature teratoma of the ovary. GCC arising within a mature teratoma of the mediastinum has not been described in the English-language literature. We report a case of this previously undescribed entity and provide a review of the literature on mediastinal teratomas with malignant transformation. The histologic findings included uniform, smooth-bordered glandular nests lined by goblet cells and admixed endocrine and Paneth cells. Occasional tubular glands were present, as were transitional type glands with both goblet cell and tubular features. Cytologic atypia was minimal, and mitotic activity was rare. Immunohistochemical studies showed positive staining of GCC tumor cells with chromogranin, cytokeratin, neuron-specific enolase, serotonin (focal), and Leu-7 (focal). The GCC component was entirely contained within the mature teratoma.

Background: Enterobiasis occurs throughout the female genital tract and may involve peritoneal surfaces. It is generally an incidental finding at surgery or at autopsy but occasionally is symptomatic. Most of the superficial lesions are composed of granulomas with variable fibrosis in which diagnostic eggs are found, often associated with degenerated adult worms. Multiple histologic sections may be required to establish the diagnosis in older lesions.Case: A case of enterobiasis of the ovary in a patient with squamous-cell carcinoma in situ of the cervix is presented. The features of enterobiasis are discussed.Conclusion: The importance of mistaking such lesions for ovarian cancer is discussed.

All living organisms face a constant barrage of DNA damage from anthropogenic and naturally occurring external and endogenous sources, yet DNA provides the blueprint for all other cellular structures. Unlike these other structural and functional molecules, DNA is not turned over in a cycle of breakdown and rebuild; DNA, by the nature of its function as an informational macromolecule and its double stranded structure, is faithfully repaired and copied to maintain the encoded information. In the case of multicellular organisms, the focus of this review, fidelity of information prevents disease, both heritable (e.g. genetic disorders), and within an individual (e.g. cancer). However, errors in DNA replication and repair serve as the grist of evolution; in some sense, these errors are essential to life as we know it. With few exceptions, the repair of DNA damage requires the action of one or more DNA polymerases. In many cases, these are specialized polymerases, recruited to the site of damage for their specific biochemical properties. In this literature review, I will present different types of DNA damage, the biochemical systems utilized in the repair of this damage, the role of various enzymes in this repair – emphasizing the role of specific DNA polymerases – and the outcome of repair – including the resulting mutation spectrum – where relevant.

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by gammaH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.

Discrete metaphyseal radiolucent lesions, often termed "metaphyseal cysts", are a frequent finding in Legg-Calvé-Perthes disease. Recent literature emphasizes that such lesions represent ectopic physeal cartilage. This paper reports a case in which biopsy of the metaphyseal lesion indicated that no cartilage was present.

The diagnosis of bacterial infections can be difficult and time consuming. Rapid and reliable molecular triage of potentially infected patients, particularly the young and the elderly, would prevent unnecessary hospitalizations, reduce associated medical costs, and improve the quality of care. Polymerase chain reaction (PCR) amplification utilizing a universal bacterial primer pair, followed by hybridization with species-specific probes, would allow rapid identification of the presence or absence of bacterial DNA, along with an identification of the bacterial species present. Molecular microbiological analyses will require access to bacterial strain standards that can be catalogued and distributed to clinical laboratories. We amplified template DNA in filter paper spots containing boiled bacteria from 14 clinical isolates using a universal primer pair for the 16S ribosomal RNA (rDNA) coding sequence. Species-specific probes were hybridized to the amplification products for bacterial species identification. We conclude that template DNA can be identified with species-specific probes after universal bacterial amplification with a single primer pair. We also demonstrate a rapid and efficient method for the long-term storage and cataloguing of bacterial DNA for use in quality control at clinical laboratories adopting molecular diagnostic methodologies. We speculate that PCR amplification combined with species-specific probe hybridization not only will represent an improvement over culture-based methods in terms of speed, sensitivity, and cost, but will also allow for the identification of unculturable bacteria and emerging or reemerging pathogenic organisms.

Monostotic Paget's disease of bone is an uncommon disease, reported in the hand in only a few instances. We have described an additional case involving a metacarpal bone in a 43-year-old man. We believe this disease should be considered in the radiological differential diagnosis of lytic expansile, subchondral lesions of the hand.

Our group and others have shown that a single, universal primer pair designed to anneal to highly conserved regions within the bacterial 16S ribosomal RNA coding sequence (16S rDNA) can amplify genomic DNA from a wide variety of bacterial organisms. Since the highly conserved regions of the 16S rDNA flank regions of the gene which vary between species, we proposed to determine whether products amplified with a universal primer pair could then be used for identification of the species with specific hybridization probes. We examined a variety of bacterial 16S rDNA sequences from GenEMBL, a non-overlapping nucleotide sequence database, using FastA (GCG, Madison, WI). These sequences were aligned using PileUp (GCG, Madison, WI) and 19-21 bp sequences were identified with apparent species specificity. PCR amplification was performed with the universal primer pair, p806R and p8FPL, and amplification products were evaluated by manual and automated methods. Dot blots were prepared with the PCR products from both Gram positive and Gram negative organisms and hybridized with end-labeled species specific probes. Using this approach we were able to differentiate between PCR products generated from different bacterial DNA templates. We conclude, that individual species can be identified by species specific sequence variability within amplification products generated following PCR with a universal bacterial primer pair. We speculate that the application of molecular genetic technology for the diagnosis of bacterial infections will reduce the cost and the time for identification of pathogens, will allow for the molecular triage of sepsis, and will improve the quality of care for patients with infections.(E.R.B. McCabe is an Academic Associate for Corning-Nichols Institute.)

Fig. 1 A-C. Chronological radiographic sequences of the fight fifth metacarpal bone during the different stages of disease. A lyric and expansile lesion within the medullary canal is seen (A, C). After treatment, a sclerosing reparative pattern is present (B) of fracture or cortical interruption. A subchondral location was noted. Enchondroma was the clinical and radiographic diagnosis. Extensive curettage of the medullary canal was performed, after which the cavity was packed with a cancellous bone graft obtained f rom the right iliac crest. During the curettage a thick fluidlike material was observed, and the bone fragments obtained were submitted for histological analysis. A radiological study performed 3 months later revealed a trabecular, sclerosing reparative pat tern (Fig. 1 B). Three years later, in January 1992, a new roentgenogram disclosed an expansile lytic lesion in the same anatomical zone, very similar radiographically to the original lesion (Fig. 1 C). Curettage was performed at that time. Periodic skeletal surveys were unremarkable except for the findings in the hand.