In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.

Outer membrane vesicles (OMVs) isolated from Salmonella Typhimurium are potentially useful for developing subunit vaccines because of high immunogenicity and protective efficacy. However, flagella might remain in OMV pellets following OMV purification, resulting in non-essential immune responses and counteraction of bacterial protective immune responses when developing a vaccine against infection of multiple serotypes Salmonella. In this study, a flagellin-deficient S. Typhimurium mutant was constructed. Lipopolysaccharide profiles, protein profiles and cryo-electron microscopy revealed that there were no significant differences between the wild-type and mutant OMVs, with the exception of a large amount of flagellin in the wild-type OMVs. Neither the wild-type OMVs nor the non-flagellin OMVs were toxic to macrophages. Mice immunized with the non-flagellin OMVs produced high concentrations of IgG. The non-flagellin OMVs elicited strong mucosal antibody responses in mice when administered via the intranasal route in addition to provoking higher cross-reactive immune responses against OMPs isolated from S. Choleraesuis and S. Enteritidis. Both intranasal and intraperitoneal immunization with the non-flagellin OMVs provided efficient protection against heterologous S. Choleraesuis and S. Enteritidis challenge. Our results indicate that the flagellin-deficient OMVs may represent a new vaccine platform that could be exploited to facilitate the production of a broadly protective vaccine.

Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells.

In eutherians, the placenta acts as a barrier and conduit at the maternal-fetal interface. Syncytiotrophoblasts, the multinucleated cells that cover the placental villous tree surfaces of the human placenta, are directly bathed in maternal blood and are formed by the fusion of progenitor cytotrophoblasts that underlie them. Despite their crucial role in fetal protection, many of the events that govern trophoblast fusion and protection from microbial infection are unknown. We describe a three-dimensional (3D)–based culture model using human JEG-3 trophoblast cells that develop syncytiotrophoblast phenotypes when cocultured with human microvascular endothelial cells. JEG-3 cells cultured in this system exhibit enhanced fusogenic activity and morphological and secretory activities strikingly similar to those of primary human syncytiotrophoblasts. RNASeq analyses extend the observed functional similarities to the transcriptome, where we observed significant overlap between syncytiotrophoblast-specific genes and 3D JEG-3 cultures. Furthermore, JEG-3 cells cultured in 3D are resistant to infection by viruses and Toxoplasma gondii, which mimics the high resistance of syncytiotrophoblasts to microbial infections in vivo. Given that this system is genetically manipulatable, it provides a new platform to dissect the mechanisms involved in syncytiotrophoblast development and microbial resistance.

We have identified a natural clay mixture that exhibits in vitro antibacterial activity against a broad spectrum of bacterial pathogens. We collected four samples from the same source and demonstrated through antibacterial susceptibility testing that these clay mixtures have markedly different antibacterial activity against Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Here, we used X-ray diffraction (XRD) and inductively coupled plasma - optical emission spectroscopy (ICP-OES) and - mass spectrometry (ICP-MS) to characterize the mineralogical and chemical features of the four clay mixture samples. XRD analyses of the clay mixtures revealed minor mineralogical differences between the four samples. However, ICP analyses demonstrated that the concentrations of many elements, Fe, Co, Cu, Ni, and Zn, in particular, vary greatly across the four clay mixture leachates. Supplementation of a non-antibacterial leachate containing lower concentrations of Fe, Co, Ni, Cu, and Zn to final ion concentrations and a pH equivalent to that of the antibacterial leachate generated antibacterial activity against E. coli and MRSA, confirming the role of these ions in the antibacterial clay mixture leachates. Speciation modeling revealed increased concentrations of soluble Cu2+ and Fe2+ in the antibacterial leachates, compared to the non-antibacterial leachates, suggesting these ionic species specifically are modulating the antibacterial activity of the leachates. Finally, linear regression analyses comparing the log(10) reduction in bacterial viability to the concentration of individual ion species revealed positive correlations with Zn2+ and Cu2+ and antibacterial activity, a negative correlation with Fe3+, and no correlation with pH. Together, these analyses further indicate that the ion concentration of specific species (Fe2+, Cu2+, and Zn2+) are responsible for antibacterial activity and that killing activity is not solely attributed to pH.

Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.

Improved tools for providing specific intraoperative diagnoses could improve patient care. In neurosurgery, intraoperatively differentiating non-operative lesions such as CNS B-cell lymphoma from operative lesions can be challenging, often necessitating immunohistochemical (IHC) procedures which require up to 24-48 hours. Here, we evaluate the feasibility of generating rapid ex vivo specific labeling using a novel lymphoma-specific fluorescent switchable aptamer. Our B-cell lymphoma-specific switchable aptamer produced only low-level fluorescence in its unbound conformation and generated an 8-fold increase in fluorescence once bound to its target on CD20-positive lymphoma cells. The aptamer demonstrated strong binding to B-cell lymphoma cells within 15 minutes of incubation as observed by flow cytometry. We applied the switchable aptamer to ex vivo xenograft tissue harboring B-cell lymphoma and astrocytoma, and within one hour specific visual identification of lymphoma was routinely possible. In this proof-of-concept study in human cell culture and orthotopic xenografts, we conclude that a fluorescent switchable aptamer can provide rapid and specific labeling of B-cell lymphoma, and that developing aptamer-based labeling approaches could simplify tissue staining and drastically reduce time to histopathological diagnoses compared with IHC-based methods. We propose that switchable aptamers could enhance expeditious, accurate intraoperative decision-making.

Improved antigenicity against HIV-1 envelope (Env) protein is needed to elicit vaccine-induced protective immunity in humans. Here we describe the first tests in non-human primates (NHPs) of Env gp140 protein fused to a humanized anti-LOX-1 recombinant antibody for delivering Env directly to LOX-1-bearing antigen presenting cells, especially dendritic cells (DC). LOX-1, or 1ectin-like oxidized low-density lipoprotein (LDL) receptor-1, is expressed on various antigen presenting cells and endothelial cells, and is involved in promoting humoral immune responses. The anti-LOX-1 Env gp140 fusion protein was tested for priming immune responses and boosting responses in animals primed with replication competent NYVAC-KC Env gp140 vaccinia virus. Anti-LOX-1 Env gp140 vaccination elicited robust cellular and humoral responses when used for either priming or boosting immunity. Co-administration with Poly ICLC, a TLR3 agonist, was superior to GLA, a TLR4 agonist. Both CD4⁺ and CD8⁺ Env-specific T cell responses were elicited by anti-LOX-1 Env gp140, but in particular the CD4⁺ T cells were multifunctional and directed to multiple epitopes. Serum IgG and IgA antibody responses induced by anti-LOX-1 Env gp140 against various gp140 domains were cross-reactive across HIV-1 clades; however, the sera neutralized only HIV-1 bearing sequences most similar to the clade C 96ZM651 Env gp140 carried by the anti-LOX-1 vehicle. These data, as well as the safety of this protein vaccine, justify further exploration of this DC-targeting vaccine approach for protective immunity against HIV-1.

Background: The evolutionary diversification of gene families through gene creation (and loss) is a dynamic process believed to be critical to the evolution of functional novelty. Previous identification of a closely related family of eight annotated metalloprotease genes of the M17 Merops family in the Drosophila sperm proteome (termed, S perm-L eucylA minoP eptidases, S-LAPs 1-8) led us to hypothesize that this gene family may have experienced such a diversification during insect evolution.

Results: To assess putative functional activities of S-LAPs, we (i) demonstrated that all S-LAPs are specifically expressed in the testis, (ii) confirmed their presence in sperm by two-dimensional gel electrophoresis and mass spectrometry, (iii) determined that they represent a major portion of the total protein in sperm and (iv) identified aminopeptidase enzymatic activity in sperm extracts using LAP-specific substrates. Functionally significant divergence at the canonical M17 active site indicates that the largest phylogenetic group of S-LAPs lost catalytic activity and likely acquired novel, as yet undetermined, functions in sperm prior to the expansion of the gene family.

Conclusions: Comparative genomic and phylogenetic analyses revealed the dramatic expansion of the S-LAP gene family during Drosophila evolution and copy number heterogeneity in the genomes of related insects. This finding, in conjunction with the loss of catalytic activity and potential neofunctionalization amongst some family members, extends empirical support for pervasive "revolving door" turnover in the evolution of reproductive gene family composition and function.

Researchers have iterated that the future of synthetic biology and biotechnology lies in novel consumer applications of crossing biology with engineering. However, if the new biology's future is to be sustainable, early and serious efforts must be made towards social sustainability. Therefore, the crux of new applications of synthetic biology and biotechnology is public understanding and acceptance. The RASVaccine is a novel recombinant design not found in nature that re-engineers a common bacteria ( Salmonella) to produce a strong immune response in humans. Synthesis of the RASVaccine has the potential to improve public health as an inexpensive, non-injectable product. But how can scientists move forward to create a dialogue of creating a 'common sense' of this new technology in order to promote social sustainability? This paper delves into public issues raised around these novel technologies and uses the RASVaccine as an example of meeting the public with a common sense of its possibilities and limitations.