MicroRNAs and cellular phenotypy

This Essay explores the notion that specialized cells have unique vulnerabilities to environmental contingencies that microRNAs help to counteract. Given the ease with which new microRNAs evolve, they may serve as ideal facilitators for the emergence of new cell types.     

Effect of the Earthworms Lumbricus terrestris and Aporrectodea caliginosa on Bacterial Diversity in Soil

Earthworms ingest large amounts of soil and have the potential to radically alter the biomass, activity, and structure of the soil microbial community. In this study, the diversity of eight bacterial groups from fresh soil, gut, and casts of the earthworms Lumbricus terrestris and Aporrectodea caliginosa were studied by single-strand conformation polymorphism (SSCP) analysis using both newly designed 16S rRNA gene-specific primer sets targeting Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Firmicutes and a conventional universal primer set for SSCP, with RNA and DNA as templates. In parallel, the study of the relative abundance of these taxonomic groups in the same samples was performed using fluorescence in situ hybridization. Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria were predominant in communities from the soil and worm cast samples. Representatives of classes Flavobacteria and Sphingobacteria (Bacteroidetes) and Pseudomonas spp. (low-abundant Gammaproteobacteria) were detected in soil and worm cast samples with conventional and taxon-targeting SSCP and through the sequence analysis of 16S rRNA clone libraries. Physiologically active unclassified Sphingomonadaceae (Alphaproteobacteria) and Alcaligenes spp. (Betaproteobacteria) also maintained their diversities during transit through the earthworm intestine and were found on taxon-targeting SSCP profiles from the soil and worm cast samples. In conclusion, our results suggest that some specific bacterial taxonomic groups maintain their diversity and even increase their relative numbers during transit through the gastrointestinal tract of earthworms.     

Label-Free Bacterial Imaging with Deep-UV-Laser-Induced Native Fluorescence

We introduce a near-real-time optical imaging method that works via the detection of the intrinsic fluorescence of life forms upon excitation by deep-UV (DUV) illumination. A DUV (<250-nm) source enables the detection of microbes in their native state on natural materials, avoiding background autofluorescence and without the need for fluorescent dyes or tags. We demonstrate that DUV-laser-induced native fluorescence can detect bacteria on opaque surfaces at spatial scales ranging from tens of centimeters to micrometers and from communities to single cells. Given exposure times of 100 μs and low excitation intensities, this technique enables rapid imaging of bacterial communities and cells without irreversible sample alteration or destruction. We also demonstrate the first noninvasive detection of bacteria on in situ-incubated environmental experimental samples from the deep ocean (Lo''ihi Seamount), showing the use of DUV native fluorescence for in situ detection in the deep biosphere and other nutrient-limited environments.Bacteria are widely recognized for living in extreme environments and as integral players in processes as varied as weathering, corrosion, environmental remediation, pathogenesis, and symbiosis (3, 4, 26). In most of these cases, surface-bound bacteria play key roles (1, 7, 19) and pose a particular challenge for researchers: the detection and imaging of life on reflective and/or fluorescent surfaces at the microbial (μm) scale (5, 12, 18). In environments ranging from the deep subsurface biosphere, dry deserts, and deep ice cores to hospitals and clean rooms, concentrations of bacteria, either as spores or active cells, can range from 10⁹ to less than 1,000 cells/gram (14, 22, 24, 25, 29, 34). Finding and quantifying these microbes when they are on surfaces usually involves epifluorescence techniques, using dyes that bind to DNA or proteins, and examining the fluorescence of those dyes under UV or visible illumination (6, 8, 9, 16, 23, 31).Current tagging methods offer a number of significant disadvantages. First, the mineral surfaces on which the microbes are found are often themselves highly fluorescent, making the microbes difficult or impossible to differentiate; second, the act of adding the fluorescent probe can alter the physical and chemical nature of the system; additionally, nonspecific binding can lead to overestimation of cell abundance (2, 18). Because of the problems associated with the fluorescence of minerals and staining to detect microbial cells, researchers typically resort to physically removing cells from surfaces and staining/counting them separately from their matrix (12). This is an inefficient process that involves both cell loss and the loss of information about the mineralogical context that may have an influence on the microbial ecology. More recently, cell staining of active cells with SYBR green 1 and a computer-assisted analysis method has demonstrated an ability to separate fluorescent cells from nonspecific binding (17). However, a label-free method to search for and quantify the distribution and abundance of bacteria on natural samples over multiple spatial scales has not been available.Label-free optical approaches using Raman scattering methods have been offered as a nondestructive imaging solution (13, 27). However, these systems utilize laser energies greater than 1 × 10⁹ joules/cm², exceeding the energies necessary for chemical damage to the cell (33), require relatively flat surfaces for optimal collection efficiency, and can suffer from background fluorescence of the target and the substrate it may reside on.In response to these challenges, we have developed an optical method that enables detection and imaging of single bacterial cells on natural and opaque surfaces and assessment of bacterial density and distribution of single cells to biofilms over spatial scales ranging from microns to centimeters. The method utilizes deep-UV (DUV) (<250-nm)-laser-induced native fluorescence of organic components intrinsic to the cell or spore while avoiding autofluorescence interference from the substrate. Here we show DUV native fluorescence as a near-real-time optical imaging method and demonstrate the first noninvasive detection of bacteria on in situ-incubated environmental experimental samples from the deep ocean (Lo''ihi Seamount) for which we correlate the bacterial biomass to distributions of the iron-oxide precipitates.     

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.     

New primers for single-copy nuclear-encoded chloroplast-expressed glutamine synthetase (ncpGS) in Oxalidaceae

? Premise of the study: New primers were developed for the nuclear marker glutamine synthetase (ncpGS) in Oxalidaceae. ? Methods and Results: New forward and reverse primers were designed and tested across a wide range of Oxalidaceae. Selected taxa were sequenced to confirm homology. Potential for phylogenetic study was assessed by comparing sequenced taxa with commonly used nuclear and plastid markers. ? Conclusions: Four out of five Oxalidaceae genera and all tested Oxalis spp. amplified successfully. Sequencing confirmed homology of the amplicon. Parsimony analysis of ncpGS showed that it is a promising candidate for future phylogenetic work in Oxalidaceae.     

Open Capture–Recapture Models with Heterogeneity: II. Jolly–Seber Model

Summary Estimation of abundance is important in both open and closed population capture–recapture analysis, but unmodeled heterogeneity of capture probability leads to negative bias in abundance estimates. This article defines and develops a suite of open population capture–recapture models using finite mixtures to model heterogeneity of capture and survival probabilities. Model comparisons and parameter estimation use likelihood‐based methods. A real example is analyzed, and simulations are used to check the main features of the heterogeneous models, especially the quality of estimation of abundance, survival, recruitment, and turnover. The two major advances in this article are the provision of realistic abundance estimates that take account of heterogenetiy of capture, and an appraisal of the amount of overestimation of survival arising from conditioning on the first capture when heterogeneity of survival is present.     

Efficient repair of DNA double-strand breaks in malignant cells with structural instability

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.