Natural Products Chemistry and Taxonomy of the Marine Cyanobacterium Blennothrix cantharidosmum

A Papua New Guinea field collection of the marine cyanobacterium Blennothrix cantharidosmum was investigated for its cytotoxic constituents. Bioassay-guided isolation defined the cytotoxic components as the known compounds lyngbyastatins 1 and 3. However, six new acyl proline derivatives, tumonoic acids D−I, plus the known tumonoic acid A were also isolated. Their planar structures were defined from NMR and MS data, while their stereostructures followed from a series of chiral chromatographies, degradation sequences, and synthetic approaches. The new compounds were tested in an array of assays, but showed only modest antimalarial and inhibition of quorum sensing activities. Nevertheless, these are the first natural products to be reported from this genus, and this inspired a detailed morphologic and 16S rDNA-based phylogenetic analysis of the producing organism

Ancient origins determine global biogeography of hot and cold desert cyanobacteria

Factors governing large-scale spatio-temporal distribution of microorganisms remain unresolved, yet are pivotal to understanding ecosystem value and function. Molecular genetic analyses have focused on the influence of niche and neutral processes in determining spatial patterns without considering the temporal scale. Here, we use temporal phylogenetic analysis calibrated using microfossil data for a globally sampled desert cyanobacterium, Chroococcidiopsis, to investigate spatio-temporal patterns in microbial biogeography and evolution. Multilocus phylogenetic associations were dependent on contemporary climate with no evidence for distance-related patterns. Massively parallel pyrosequencing of environmental samples confirmed that Chroococcidiopsis variants were specific to either hot or cold deserts. Temporally scaled phylogenetic analyses showed no evidence of recent inter-regional gene flow, indicating populations have not shared common ancestry since before the formation of modern continents. These results indicate that global distribution of desert cyanobacteria has not resulted from widespread contemporary dispersal but is an ancient evolutionary legacy. This highlights the importance of considering temporal scales in microbial biogeography

TROP2 Expressed in the Trunk of the Ureteric Duct Regulates Branching Morphogenesis during Kidney Development

TROP2, a cell surface protein structurally related to EpCAM, is expressed in various carcinomas, though its function remains largely unknown. We examined the expression of TROP2 and EpCAM in fetal mouse tissues, and found distinct patterns in the ureteric bud of the fetal kidney, which forms a tree-like structure. The tip cells in the ureteric bud proliferate to form branches, whereas the trunk cells differentiate to form a polarized ductal structure. EpCAM was expressed throughout the ureteric bud, whereas TROP2 expression was strongest at the trunk but diminished towards the tips, indicating the distinct cell populations in the ureteric bud. The cells highly expressing TROP2 (TROP2high) were negative for Ki67, a proliferating cell marker, and TROP2 and collagen-I were co-localized to the basal membrane of the trunk cells. TROP2high cells isolated from the fetal kidney failed to attach and spread on collagen-coated plates. Using MDCK cells, a well-established model for studying the branching morphogenesis of the ureteric bud, TROP2 was shown to inhibit cell spreading and motility on collagen-coated plates, and also branching in collagen-gel cultures, which mimic the ureteric bud's microenvironment. These results together suggest that TROP2 modulates the interaction between the cells and matrix and regulates the formation of the ureteric duct by suppressing branching from the trunk during kidney development

Claudin-7 Is Frequently Overexpressed in Ovarian Cancer and Promotes Invasion

Background: Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC. Methodology/Principal Findings: A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration

Non-Invasive Quantification of White and Brown Adipose Tissues and Liver Fat Content by Computed Tomography in Mice

OBJECTIVES: Obesity and its distribution pattern are important factors for the prediction of the onset of diabetes in humans. Since several mouse models are suitable to study the pathophysiology of type 2 diabetes the aim was to validate a novel computed tomograph model (Aloka-Hitachi LCT-200) for the quantification of visceral, subcutaneous, brown and intrahepatic fat depots in mice. METHODS: Different lean and obese mouse models (C57BL/6, B6.V-Lep(ob), NZO) were used to determine the most adequate scanning parameters for the detection of the different fat depots. The data were compared with those obtained after preparation and weighing the fat depots. Liver fat content was determined by biochemical analysis. RESULTS: The correlations between weights of fat tissues on scale and weights determined by CT were significant for subcutaneous (r(2) = 0.995), visceral (r(2) = 0.990) and total white adipose tissue (r(2) = 0.992). Moreover, scans in the abdominal region, between lumbar vertebrae L4 to L5 correlated with whole-body fat distribution allowing experimenters to reduce scanning time and animal exposure to radiation and anesthesia. Test-retest reliability and measurements conducted by different experimenters showed a high reproducibility in the obtained results. Intrahepatic fat content estimated by CT was linearly related to biochemical analysis (r(2) = 0.915). Furthermore, brown fat mass correlated well with weighted brown fat depots (r(2) = 0.952). In addition, short-term cold-expose (4 °C, 4 hours) led to alterations in brown adipose tissue attributed to a reduction in triglyceride content that can be visualized as an increase in Hounsfield units by CT imaging. CONCLUSION: The 3D imaging of fat by CT provides reliable results in the quantification of total, visceral, subcutaneous, brown and intrahepatic fat in mice. This non-invasive method allows the conduction of longitudinal studies of obesity in mice and therefore enables experimenters to investigate the onset of complex diseases such as diabetes and obesity

Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes

CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B′) as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B′ genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B′, but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as “viritopes” to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities

Sequence Diversities of Serine-Aspartate Repeat Genes among Staphylococcus aureus Isolates from Different Hosts Presumably by Horizontal Gene Transfer

BACKGROUND: Horizontal gene transfer (HGT) is recognized as one of the major forces for bacterial genome evolution. Many clinically important bacteria may acquire virulence factors and antibiotic resistance through HGT. The comparative genomic analysis has become an important tool for identifying HGT in emerging pathogens. In this study, the Serine-Aspartate Repeat (Sdr) family has been compared among different sources of Staphylococcus aureus (S. aureus) to discover sequence diversities within their genomes. METHODOLOGY/PRINCIPAL FINDINGS: Four sdr genes were analyzed for 21 different S. aureus strains and 218 mastitis-associated S. aureus isolates from Canada. Comparative genomic analyses revealed that S. aureus strains from bovine mastitis (RF122 and mastitis isolates in this study), ovine mastitis (ED133), pig (ST398), chicken (ED98), and human methicillin-resistant S. aureus (MRSA) (TCH130, MRSA252, Mu3, Mu50, N315, 04-02981, JH1 and JH9) were highly associated with one another, presumably due to HGT. In addition, several types of insertion and deletion were found in sdr genes of many isolates. A new insertion sequence was found in mastitis isolates, which was presumably responsible for the HGT of sdrC gene among different strains. Moreover, the sdr genes could be used to type S. aureus. Regional difference of sdr genes distribution was also indicated among the tested S. aureus isolates. Finally, certain associations were found between sdr genes and subclinical or clinical mastitis isolates. CONCLUSIONS: Certain sdr gene sequences were shared in S. aureus strains and isolates from different species presumably due to HGT. Our results also suggest that the distributional assay of virulence factors should detect the full sequences or full functional regions of these factors. The traditional assay using short conserved regions may not be accurate or credible. These findings have important implications with regard to animal husbandry practices that may inadvertently enhance the contact of human and animal bacterial pathogens

CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

BACKGROUND: Endothelial E-selectin has been shown to play a pivotal role in mediating cell-cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. However, the counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: By assessing migration of various breast cancer cells across TNF-alpha pre-activated human umbilical vein endothelial cells (HUVECs), we found that breast cancer cells migrated across HUVEC monolayers differentially and that transmigration was E-selectin dependent. Cell surface labeling with the E-selectin extracellular domain/Fc chimera (exE-selectin/Fc) showed that the transmigration capacity of breast cancer cells was correlated to both the expression level and localization pattern of E-selectin binding protein(s) on the tumor cell surface. The exE-selectin/Fc strongly bound to metastatic MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells, but not non-metastatic MCF-7 and T47D cells. Binding of exE-selectin/Fc was abolished by removal of tumor cell surface sialyl lewis x (sLe(x)) moieties. Employing an exE-selectin/Fc affinity column, we further purified the counterreceptor of E-selectin from metastatic breast cancer cells. The N-terminal protein sequence and cDNA sequence identified this E-selectin ligand as a approximately 170 kD human CD44 variant 4 (CD44v4). Purified CD44v4 showed a high affinity for E-selectin via sLe(x) moieties and, as expected, MDA-MB-231 cell adhesion to and migration across HUVEC monolayers were significantly reduced by down-regulation of tumor cell CD44v4 via CD44v4-specific siRNA. CONCLUSIONS/SIGNIFICANCE: We demonstrated, for the first time, that breast cancer cell CD44v4 is a major E-selectin ligand in facilitating tumor cell migration across endothelial monolayers. This finding offers new insights into the molecular basis of E-selectin-dependent adhesive interactions that mediate breast cancer cell transendothelial metastasis

Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis

BACKGROUND: Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species. The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes. The bacterium Atopobium vaginae has been recognized only recently. It is not readily identified by commercial diagnostic kits. Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess. METHODS: Nucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp. predominant; Nugent score ≤ 3) and abnormal flora (Nugent score ≥ 4). These women ranged in age from 14 to 48 and 82% were African American. RESULTS: The DGGE banding patterns of normal and BV-positive patients were recognizably distinct. Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane. More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients. (P < 0.001) Two A. vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism. Two A. vaginae 16S rRNA gene sequences were identified among the clinical isolates. The same two sequences were obtained from DGGE bands of the corresponding vaginal flora. The sequences differed by one nucleotide over the short (~300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels. Both isolates were strict anaerobes and highly metronidazole resistant. CONCLUSION: The results suggest that A. vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora. This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant