The metastatic activity of breast carcinomas results from complex genetic changes in epithelial tumor cells and accounts for 90% of deaths in affected patients. Although the invasion of the local lymphatic vessels and veins by malignant breast tumor cells and their subsequent metastasis to the lung, has been recognized, the mechanisms behind the metastatic activity of breast tumor cells to other distal organs and the pathogenesis of metastatic cancer are not well understood. In this study, we utilized derivatives of the well-established and highly metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231 (MDA-231) to study breast tumor metastasis in a mouse model. These MDA-231 derivatives had depleted expression of Kaiso, a POZ-ZF transcription factor that is highly expressed in malignant, triple negative breast cancers. We previously reported that Kaiso depletion attenuates the metastasis of xenografted MDA-231 cells. Herein, we describe the pathological features of the metastatic activity of parental (Kaiso^positive) versus Kaiso^depleted MDA-231 cells. Both Kaiso^positive and Kaiso^depleted MDA-231 cells metastasized from the original tumor in the mammary fat pad to the lung. However, while Kaiso^positive cells formed large masses in the lung parenchyma, invaded large pulmonary blood vessels and formed secondary metastases and large tumors in the distal organs, Kaiso^depleted cells metastasized only to the lung where they formed small metastatic lesions. Importantly, intravascular invasion and secondary metastases in distal organs were not observed in mice xenografted with Kaiso^depleted cells. It thus appears that the lung may constitute a barrier for less invasive breast tumors such as the Kaiso^depleted TNBC cells; this barrier may limit tumor growth and prevents Kaiso^depleted TNBC cells from invading the pulmonary blood vessels and forming secondary metastases in distal organs.

The relationship between sequence and binding properties of an aptamer for immunoglobulin E (IgE) was investigated using custom DNA microarrays. Single, double and some triple mutations of the aptamer sequence were created to evaluate the importance of specific base composition on aptamer binding. The majority of the positions in the aptamer sequence were found to be immutable, with changes at these positions resulting in more than a 100-fold decrease in binding affinity. Improvements in binding were observed by altering the stem region of the aptamer, suggesting that it plays a significant role in binding. Results obtained for the various mutations were used to estimate the information content and the probability of finding a functional aptamer sequence by selection from a random library. For the IgE-binding aptamer, this probability is on the order of 10^-10 to 10^-9. Results obtained for the double and triple mutations also show that there are no compensatory mutations within the space defined by those mutations. Apparently, at least for this particular aptamer, the functional sequence space can be represented as a rugged landscape with sharp peaks defined by highly constrained base compositions. This makes the rational optimization of aptamer sequences using step-wise mutagenesis approaches very challenging.

The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743–D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease.

Low fluid shear force, including that encountered in microgravity models, induces bacterial responses, but the range of bacteria capable of responding to this signal remains poorly characterized. We systematically analyzed a range of Gram negative Enterobacteriaceae for conservation of the low-shear modeled microgravity (LSMMG) response using phenotypic assays, qPCR, and targeted mutations. Our results indicate LSMMG response conservation across Enterobacteriacae with potential variance in up- or down-regulation of a given response depending on genus. Based on the data, we analyzed the role of the trp operon genes and the TrpR regulator in the LSMMG response using targeted mutations in these genes in S. Typhimurium and E. coli. We found no alteration of the LSMMG response compared to WT in these mutant strains under the conditions tested here. To our knowledge, this study is first-of-kind for Citrobacter, Enterobacter, and Serratia, presents novel data for Escherichia, and provides the first analysis of trp genes in LSMMG responses. This impacts our understanding of how LSMMG affects bacteria and our ability to modify bacteria with this condition in the future.

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3′untranslated region (3′UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3′UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3′UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis.

The Florence Statement on Triclosan and Triclocarban documents a consensus of more than 200 scientists and medical professionals on the hazards of and lack of demonstrated benefit from common uses of triclosan and triclocarban. These chemicals may be used in thousands of personal care and consumer products as well as in building materials. Based on extensive peer-reviewed research, this statement concludes that triclosan and triclocarban are environmentally persistent endocrine disruptors that bioaccumulate in and are toxic to aquatic and other organisms. Evidence of other hazards to humans and ecosystems from triclosan and triclocarban is presented along with recommendations intended to prevent future harm from triclosan, triclocarban, and antimicrobial substances with similar properties and effects. Because antimicrobials can have unintended adverse health and environmental impacts, they should only be used when they provide an evidence-based health benefit. Greater transparency is needed in product formulations, and before an antimicrobial is incorporated into a product, the long-term health and ecological impacts should be evaluated.

Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis.

The brain is a common site of metastatic disease in patients with breast cancer, which has few therapeutic options and dismal outcomes. The purpose of our study was to identify common and rare events that underlie breast cancer brain metastasis. We performed deep genomic profiling, which integrated gene copy number, gene expression and DNA methylation datasets on a collection of breast brain metastases. We identified frequent large chromosomal gains in 1q, 5p, 8q, 11q, and 20q and frequent broad-level deletions involving 8p, 17p, 21p and Xq. Frequently amplified and overexpressed genes included ATAD2, BRAF, DERL1, DNMTRB and NEK2A. The ATM, CRYAB and HSPB2 genes were commonly deleted and underexpressed. Knowledge mining revealed enrichment in cell cycle and G2/M transition pathways, which contained AURKA, AURKB and FOXM1. Using the PAM50 breast cancer intrinsic classifier, Luminal B, Her2+/ER negative, and basal-like tumors were identified as the most commonly represented breast cancer subtypes in our brain metastasis cohort. While overall methylation levels were increased in breast cancer brain metastasis, basal-like brain metastases were associated with significantly lower levels of methylation. Integrating DNA methylation data with gene expression revealed defects in cell migration and adhesion due to hypermethylation and downregulation of PENK, EDN3, and ITGAM. Hypomethylation and upregulation of KRT8 likely affects adhesion and permeability. Genomic and epigenomic profiling of breast brain metastasis has provided insight into the somatic events underlying this disease, which have potential in forming the basis of future therapeutic strategies.

Anode-respiring bacteria (ARB) generate electric current in microbial electrochemical cells (MXCs) by channeling electrons from the oxidation of organic substrates to an electrode. Production of high current densities by monocultures in MXCs has resulted almost exclusively from the activity of Geobacter sulfurreducens, a neutrophilic freshwater Fe(III)-reducing bacterium and the highest-current-producing member documented for the Geobacteraceae family of the Deltaproteobacteria. Here we report high current densities generated by haloalkaliphilic Geoalkalibacter spp., thus broadening the capability for high anode respiration rates by including other genera within the Geobacteraceae. In this study, acetate-fed pure cultures of two related Geoalkalibacter spp. produced current densities of 5.0 to 8.3 and 2.4 to 3.3 A m^-2 under alkaline (pH 9.3) and saline (1.7% NaCl) conditions, respectively. Chronoamperometric studies of halophilic Glk. subterraneus DSM 23483 and alkaliphilic Glk. ferrihydriticus DSM 17813 suggested that cells performed long-range electron transfer through electrode-attached biofilms and not through soluble electron shuttles. Glk. ferrihydriticus also oxidized ethanol directly to produce current, with maximum current densities of 5.7 to 7.1 A m^-2 and coulombic efficiencies of 84 to 95%. Cyclic voltammetry (CV) elicited a sigmoidal response with characteristic onset, midpoint, and saturation potentials, while CV performed in the absence of an electron donor suggested the involvement of redox molecules in the biofilm that were limited by diffusion. These results matched those previously reported for actively respiring Gb. sulfurreducens biofilms producing similar current densities (~5 to 9 A m^-2).

Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo. We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 10[superscript 5] CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 10[superscript 9] CFU of wild-type S. Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.