People in developed countries spend approximately 90% of their lives indoors, yet we know little about the source and diversity of microbes in built environments. In this study, we combined culture-based cell counting and multiplexed pyrosequencing of environmental ribosomal RNA (rRNA) gene sequences to investigate office space bacterial diversity in three metropolitan areas. Five surfaces common to all offices were sampled using sterile double-tipped swabs, one tip for culturing and one for DNA extraction, in 30 different offices per city (90 offices, 450 total samples). 16S rRNA gene sequences were PCR amplified using bar-coded “universal” bacterial primers from 54 of the surfaces (18 per city) and pooled for pyrosequencing. A three-factorial Analysis of Variance (ANOVA) found significant differences in viable bacterial abundance between offices inhabited by men or women, among the various surface types, and among cities. Multiplex pyrosequencing identified more than 500 bacterial genera from 20 different bacterial divisions. The most abundant of these genera tended to be common inhabitants of human skin, nasal, oral or intestinal cavities. Other commonly occurring genera appeared to have environmental origins (e.g., soils). There were no significant differences in the bacterial diversity between offices inhabited by men or women or among surfaces, but the bacterial community diversity of the Tucson samples was clearly distinguishable from that of New York and San Francisco, which were indistinguishable. Overall, our comprehensive molecular analysis of office building microbial diversity shows the potential of these methods for studying patterns and origins of indoor bacterial contamination. “[H]umans move through a sea of microbial life that is seldom perceived except in the context of potential disease and decay.” – Feazel et al. (2009).

Infants in Neonatal Intensive Care Units (NICUs) are particularly susceptible to opportunistic infection. Infected infants have high mortality rates, and survivors often suffer life-long neurological disorders. The causes of many NICU infections go undiagnosed, and there is debate as to the importance of inanimate hospital environments (IHEs) in the spread of infections. We used culture-independent next-generation sequencing to survey bacterial diversity in two San Diego NICUs and to track the sources of microbes in these environments. Thirty IHE samples were collected from two Level-Three NICU facilities. We extracted DNA from these samples and amplified the bacterial small subunit (16S) ribosomal RNA gene sequence using ‘universal’ barcoded primers. The purified PCR products were pooled into a single reaction for pyrosequencing, and the data were analyzed using QIIME. On average, we detected 93+/−39 (mean +/− standard deviation) bacterial genera per sample in NICU IHEs. Many of the bacterial genera included known opportunistic pathogens, and many were skin-associated (e.g., Propionibacterium). In one NICU, we also detected fecal coliform bacteria (Enterobacteriales) in a high proportion of the surface samples. Comparison of these NICU-derived sequences to previously published high-throughput 16S rRNA amplicon studies of other indoor environments (offices, restrooms and healthcare facilities), as well as human- and soil-associated environments, found the majority of the NICU samples to be similar to typical building surface and air samples, with the notable exception of the IHEs which were dominated by Enterobacteriaceae. Our findings provide evidence that NICU IHEs harbor a high diversity of human-associated bacteria and demonstrate the potential utility of molecular methods for identifying and tracking bacterial diversity in NICUs.

Anthrax lethal toxin assembles at the surface of mammalian cells when the lethal factor (LF) binds via its amino-terminal domain, LF N , to oligomeric forms of activated protective antigen (PA). LF⅐PA complexes are then trafficked to acidified endosomes, where PA forms heptameric pores in the bounding membrane and LF translocates through these pores to the cytosol. We used enhanced peptide amide hydrogen/deuterium exchange mass spectrometry and directed mutagenesis to define the surface on LF N that interacts with PA. A continuous surface encompassing one face of LF N became protected from deuterium exchange when LF N was bound to a PA dimer. Directed mutational analysis demonstrated that residues within this surface on LF N interact with Lys-197 on two PA subunits simultaneously, thereby showing that LF N spans the PA subunit:subunit interface and explaining why heptameric PA binds a maximum of three LF N molecules. Our results elucidate the structural basis for anthrax lethal toxin assembly and may be useful in developing drugs to block toxin action.Bacillus anthracis, the causative agent of anthrax, secretes three monomeric proteins that assemble at the host cell surface to form a series of toxic, non-covalent complexes. Toxin assembly begins when one of these proteins, termed protective antigen (PA 83 ) 4 (83 kDa), binds to either of two cell-surface receptors (1, 2) and a 20-kDa piece is proteolytically removed from the N terminus (3). The remaining receptorbound fragment, PA 63 (63 kDa), then spontaneously self-assembles into a ring-shaped homoheptamer (PA 63 ) 7 , termed the pre-pore (4). The latter can bind up to three copies of either, or both, of the two remaining toxin components, edema factor (EF) and lethal factor (LF) (5). The combination of LF and PA is sometimes called lethal toxin, and the combination of EF and LF, edema toxin. LF and EF bind competitively to oligomeric forms of PA 63 via their homologous N-terminal ϳ250-residue domains, termed LF N and EF N (6, 7). The resulting toxic complexes are then internalized into acidified compartments (8) where the prepore converts into a membrane-spanning pore (9), allowing EF and LF to translocate to the cytosol (10, 11). Once in the cytosol, LF, a Zn 2ϩ -dependent protease (12), and EF, a calmodulin-dependent adenylate cyclase (13), exert their toxic effects by modifying specific cytosolic targets.Describing how the three components of anthrax toxin recognize each other at the molecular level can elucidate the self-assembly process at the cell surface and how they dissociate prior to, or during, translocation in the endosome. Such information could also aid in designing inhibitors that block toxin action. Previously, conservation-guided mutagenesis was used to screen for residues on LF/EF and PA that mediate complex assembly (14 -19). Lacy et al. (14) identified a small rectangular patch of seven residues on the surface of LF N /EF N that were critical for interacting with PA. However, later mutagenesis studies revealed a larger footprint on PA t...

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We investigated the effects of a laboratory curriculum developed using the socio-­scientific issues (SSI) framework to contextualize scientific and socially relevant issues for students. Using self-determination theory and hierarchical linear modeling, we examined the effects of the SSI curriculum relative to a control curriculum on student motivation in a large introductory biology course for life science majors. The SSI group had a significant increase in motivation for engaging in the laboratory work relative to motivation of the control group. Additionally, the SSI group showed higher levels of more autonomous forms of regulation concerning participation in laboratory tasks compared with the control group. Interestingly, the SSI-based curriculum seemed to have a buffering effect on typically observed decreases in student motivation over the course of a term. This buffering effect could potentially indicate greater self-determination in students experiencing an SSI-based curriculum, which could lead to greater student success and persistence. Qualitative data suggest that this increased motivation of the SSI group relative to the control group is due to enhanced feelings of relatedness experienced by students, likely due to the SSI.

Protein-protein interactions between SHEP and Cas proteins influence cellular signaling through tyrosine kinases, as well as integrin-mediated signaling, and may be linked to antiestrogen resistance. Data from past studies suggests that association between SHEP and Cas proteins is critical for these cellular effects. In this study, the interacting domains of each protein were co-expressed in bacteria and a soluble stable complex was purified. Deuterium exchange mass spectrometry was used to define regions that are buried when SHEP1 is in complex with Cas. The results reveal four segments in SHEP1 that are highly protected, including a region (residues 619-640) that contains a key residue, tyrosine 635, required for association with Cas. This region is predominately hydrophilic, yet remains protected from solvent in the complex.

Recent reform initiatives in undergraduate biology call for curricula that prepare students for dealing with real-world issues and making important links between science and society. In response to this call, we have developed an issues-based laboratory module that uses guided inquiry to integrate the concepts of animal behavior and population biology into an issue of both local and global relevance. The issue associated with this module is "What should be done about invasive crayfish?" Students investigate plausible reasons why crayfish are often successful invasive species through hypothesis testing, collection of behavioral data on live crayfish, and quantitative reasoning. Students also consider economic and environmental impacts of invasive species on local and global ecosystems. We implemented this module in a large introductory biology course and conducted survey research to evaluate the module's potential to serve as an interesting and valuable learning experience for undergraduate biology students. I n v e s t I g a t I o n Laboratory: The Behavior of Red Swamp Crayfish as an Invasive Species the winners of agonistic interactions between invasive Procambarus clarkii (red swamp crayfish) and the implications for invasive-species management. Students learn concepts and principles of animal behavior while applying this information to a real-world issue. Crayfish as Invasive-Species Models J J JRed swamp crayfish was an advantageous study species for these topics because of its value as a model for both ecological invasion and behavior. Specifically, we chose crayfish because they provide an excellent example of a locally relevant ecological issue. Given the cosmopolitan nature of their role as invaders, this module will have local relevance in a great many other places and can be adapted to many geographic areas. It is appropriate for both secondary and postsecondary biology courses.Additionally, while several hypotheses exist regarding the mechanisms that facilitate the exclusion of native species by invasive crayfish, there is agreement that agonistic (aggressive and submissive) interactions between and within species play a large role in the exclusion of native species (Gherardi & Daniels, 2004; Gherardi, 2007). Agonistic behaviors are well documented, reliable, and easy to observe in a laboratory setting (Tierney et al., 2000), making them ideal for students who lack experience with behavioral analyses. Finally, crayfish are readily available from distributors and easy to maintain in a laboratory setting. Table 2 outlines the materials needed for instructors to successfully run the laboratory module in a large or small course. Materials J J J Methods J J J