Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. Download video file.^{(57M, mp4)}     

A novel proteomic coculture model of prostate cancer cell growth

Chemotherapy and androgen ablation therapy are only temporarily effective against prostate cancer, and current studies are ongoing to test agents that target proteins responsible for autocrine and paracrine stimulated growth. Given limitations of current laboratory models to test the effect of these agents on cell growth and protein targets, we developed a coculture model that can distinguish paracrine stimulated growth and effects on proteins. We found that LNCaP prostate cancer cells and an immortalized rat prostate cell line transfected to overexpress the antiapoptotic resistance protein Bcl-2 were stimulated to grow (>2-fold increase, p < 0.01) through autocrine effects from additional cells in an upper chamber of our system. Using a proteomic approach with a two-dimensional differential in gel electrophoresis method to increase fidelity, four proteins were found to increase after autocrine induced growth stimulation. These proteins were all identified by mass spectrometry as enzymes in the glycolytic pathway, validating the ability of this system to detect both clonogenic growth and the effect on proteins. These data, therefore, demonstrate a novel coculture model for further study of agents that target proteins in pathways of paracrine or autocrine stimulated cell growth.     

The coverage and frequency of mass drug administration required to eliminate persistent transmission of soil-transmitted helminths

A combination of methods, including mathematical model construction, demographic plus epidemiological data analysis and parameter estimation, are used to examine whether mass drug administration (MDA) alone can eliminate the transmission of soil-transmitted helminths (STHs). Numerical analyses suggest that in all but low transmission settings (as defined by the magnitude of the basic reproductive number, R₀), the treatment of pre-school-aged children (pre-SAC) and school-aged children (SAC) is unlikely to drive transmission to a level where the parasites cannot persist. High levels of coverage (defined as the fraction of an age group effectively treated) are required in pre-SAC, SAC and adults, if MDA is to drive the parasite below the breakpoint under which transmission is eliminated. Long-term solutions to controlling helminth infections lie in concomitantly improving the quality of the water supply, sanitation and hygiene (WASH). MDA, however, is a very cost-effective tool in long-term control given that most drugs are donated free by the pharmaceutical industry for poor regions of the world. WASH interventions, by lowering the basic reproductive number, can facilitate the ability of MDA to interrupt transmission.     

Genetically related Clostridium difficile from water sources and human CDI cases revealed by whole-genome sequencing

Clostridium difficile isolates from the environment are closely related to those from humans, indicating a possible environmental transmission route for C. difficile infection (CDI). In this study, C. difficile was isolated from 47.3% (53/112) of lake/pond, 23.0% (14/61) of river, 20.0% (3/15) of estuary and 0.0% (0/89) of seawater samples. The most common toxigenic strain isolated was C. difficile PCR ribotype (RT) 014/020 (10.5%, 8/76). All water isolates were susceptible to fidaxomicin, metronidazole, rifaximin, amoxicillin/clavulanic acid, moxifloxacin and tetracycline. Resistance to vancomycin, clindamycin, erythromycin and meropenem was detected in 5.3% (4/76), 26.3% (20/76), 1.3% (1/76) and 6.6% (5/76) of isolates, respectively. High-resolution core-genome analysis was performed on RT 014/020 isolates of water origin and 26 clinical RT 014/020 isolates from the same year and geographical location. Notably, both human and water strains were intermixed across three sequence types (STs), 2, 13 and 49. Six closely related groups with ≤10 core-genome single nucleotide polymorphisms were identified, five of which comprised human and water strains. Overall, 19.2% (5/26) of human strains shared a recent genomic relationship with one or more water strains. This study supports the growing hypothesis that environmental contamination by C. difficile plays a role in CDI transmission.     

Prospects for Bonobo Insectivory: Lui Kotal, Democratic Republic of Congo

Chimpanzees (Pan troglodytes) are well-known to eat invertebrates, especially social insects, across Africa, but allopatric bonobos (P. paniscus) are not. Bonobo insectivory is sparsely documented and apparently sporadic. However, the availability to bonobos of social insect prey and raw materials with which to make tools to exploit them is unknown. Here, we test a set of hypotheses that relates to questions of presence, abundance, density, and distribution of taxa that Pan consume and of vegetation suitable for making extractive foraging tools. We worked at Lui Kotal, Democratic Republic of Congo, where unprovisioned bonobos live in intact forest, far from villages. We collected insect and fecal specimens, transected for prey and assessed raw materials, and monitored mounds of Macrotermes. All but 1 of the major taxa of relevant termites, ants, and (stinging) honey bees were present. The 3 main taxa of insects that chimpanzees elsewhere eat —Macrotermes (fungus-growing termites), Dorylus (Anomma; army or driver ants), and Apis (honey bees)— were abundant and widespread, and usually at densities exceeding those at well-known chimpanzee study-sites. Similarly, woody and nonwoody vegetation suitable for making fishing probes was common at mounds of Macrotermes. There is no obvious ecological reason why bonobos should not use elementary technology in extractive foraging, e.g., termite-fish, ant-fish, ant-dip, honey-dip, to obtain social insects.     

Integrin alpha6beta4-erbB2 complex inhibits haptotaxis by up-regulating E-cadherin cell-cell junctions in keratinocytes

Keratinocyte integrins alpha6beta4 and alpha3beta1 bind laminin-5, a component of basement membranes. We previously demonstrated that in keratinocytes, haptotactic migration on laminin-5 was stimulated by anti-beta1 integrin-activating antibody TS2/16, whereas antibodies to alpha6 and beta4, respectively, blocked TS2/16-induced, alpha3beta1-dependent migration. Moreover, alpha6beta4-associated haptotaxis inhibition was linked to a phosphatidylinositol 3-kinase (PI3K) pathway and required erbB2 activation. erbB2, the ligand-less member of the epidermal growth factor receptor family, was shown to form a complex with the hemidesmosomal integrin alpha6beta4. Here, we demonstrate that alpha6beta4 inhibitory effects on haptotaxis are abolished by an anti-E-cadherin antibody, which interferes with cell-cell adhesion. Furthermore, antibodies to alpha6 and beta4 stimulated adhesion to an E-cadherin-Fc recombinant protein. In addition, anti-alpha6/beta4 antibodies increased colony size in plated cells, stimulated cell-cell aggregation, and up-regulated E-cadherin localization to cell-cell contacts. These effects were abolished when erbB2 or PI3K were blocked. These results indicate that stimulation of alpha6beta4 increases E-cadherin-mediated cell-cell adhesion and that this mechanism depends on erbB2 activation. The molecule that links alpha6beta4 with E-cadherin may be the small GTPase cdc42, an effector of PI3K, because dominant-negative cdc42 abolished the inhibitory effect of anti-alpha6/beta4 antibodies and increased basal migration, whereas constitutively active cdc42 prevented the TS2/16-induced increase in haptotaxis. These findings suggest a model whereby alpha6beta4 can augment cell-cell adhesion and slow down haptotaxis over laminin-5 and point to the alpha6beta4-erbB2 heterodimer as an important signaling complex for the formation of cohesive keratinocyte layers.     

Microbial Community Diversity of Moonmilk Deposits at Ballynamintra Cave, Co. Waterford, Ireland

Caves are extreme and specialised habitats for terrestrial life that sometimes contain moonmilk, a fine-grained paste-like secondary mineral deposit that is found in subterranean systems worldwide. While previous studies have investigated the possible role of microorganisms in moonmilk precipitation, the microbial community ecology of moonmilk deposits is poorly understood. Bacterial and fungal community structure associated with four spatially isolated microcrystalline, acicular calcite moonmilk deposits at Ballynamintra Cave (S. Ireland) was investigated during this study. Statistical analyses revealed significant differences in microbial activity, number of bacterial species, bacterial richness and diversity, and fungal diversity (Shannon's diversity) among the moonmilk sites over an area of approximately 2.5 m². However, the number of fungal species and fungal community richness were unaffected by sampling location. SIMPER analysis revealed significant differences in bacterial and fungal community composition among the sampling sites. These data suggest that a rich assemblage of microorganisms exists associated with moonmilk, with some spatial diversity, which may reflect small-scale spatial differences in cave biogeochemistry.