High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.     

An emperor penguin population estimate: the first global, synoptic survey of a species from space

Our aim was to estimate the population of emperor penguins (Aptenodytes fosteri) using a single synoptic survey. We examined the whole continental coastline of Antarctica using a combination of medium resolution and Very High Resolution (VHR) satellite imagery to identify emperor penguin colony locations. Where colonies were identified, VHR imagery was obtained in the 2009 breeding season. The remotely-sensed images were then analysed using a supervised classification method to separate penguins from snow, shadow and guano. Actual counts of penguins from eleven ground truthing sites were used to convert these classified areas into numbers of penguins using a robust regression algorithm.We found four new colonies and confirmed the location of three previously suspected sites giving a total number of emperor penguin breeding colonies of 46. We estimated the breeding population of emperor penguins at each colony during 2009 and provide a population estimate of ~238,000 breeding pairs (compared with the last previously published count of 135,000-175,000 pairs). Based on published values of the relationship between breeders and non-breeders, this translates to a total population of ~595,000 adult birds.There is a growing consensus in the literature that global and regional emperor penguin populations will be affected by changing climate, a driver thought to be critical to their future survival. However, a complete understanding is severely limited by the lack of detailed knowledge about much of their ecology, and importantly a poor understanding of their total breeding population. To address the second of these issues, our work now provides a comprehensive estimate of the total breeding population that can be used in future population models and will provide a baseline for long-term research.     

Estimating the impact of newly arrived foreign-born persons on tuberculosis in the United States

Background

Among approximately 163.5 million foreign-born persons admitted to the United States annually, only 500,000 immigrants and refugees are required to undergo overseas tuberculosis (TB) screening. It is unclear what extent of the unscreened nonimmigrant visitors contributes to the burden of foreign-born TB in the United States.

Methodology/Principal Findings

We defined foreign-born persons within 1 year after arrival in the United States as “newly arrived”, and utilized data from U.S. Department of Homeland Security, U.S. Centers for Disease Control and Prevention, and World Health Organization to estimate the incidence of TB among newly arrived foreign-born persons in the United States. During 2001 through 2008, 11,500 TB incident cases, including 291 multidrug-resistant TB incident cases, were estimated to occur among 20,989,738 person-years for the 1,479,542,654 newly arrived foreign-born persons in the United States. Of the 11,500 estimated TB incident cases, 41.6% (4,783) occurred among immigrants and refugees, 36.6% (4,211) among students/exchange visitors and temporary workers, 13.8% (1,589) among tourists and business travelers, and 7.3% (834) among Canadian and Mexican nonimmigrant visitors without an I-94 form (e.g., arrival-departure record). The top 3 newly arrived foreign-born populations with the largest estimated TB incident cases per 100,000 admissions were immigrants and refugees from high-incidence countries (e.g., 2008 WHO-estimated TB incidence rate of ≥100 cases/100,000 population/year; 235.8 cases/100,000 admissions, 95% confidence interval [CI], 228.3 to 243.3), students/exchange visitors and temporary workers from high-incidence countries (60.9 cases/100,000 admissions, 95% CI, 58.5 to 63.3), and immigrants and refugees from medium-incidence countries (e.g., 2008 WHO-estimated TB incidence rate of 15–99 cases/100,000 population/year; 55.2 cases/100,000 admissions, 95% CI, 51.6 to 58.8).

Conclusions/Significance

Newly arrived nonimmigrant visitors contribute substantially to the burden of foreign-born TB in the United States. To achieve the goals of TB elimination, direct investment in global TB control and strategies to target nonimmigrant visitors should be considered.     

Proposal for a method to estimate nutrient shock effects in bacteria

ABSTRACT: BACKGROUND: Plating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested. FINDINGS: To estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA). The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon. CONCLUSIONS: This work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration.     

Mycorrhizal‐mediated nitrogen acquisition in switchgrass under elevated temperatures and N enrichment

Arbuscular mycorrhizal fungi (AMF) can perform key roles in ecosystem functioning through improving host nutrient acquisition. Nitrogen (N) is an essential nutrient for plant growth, however, anthropogenic N loading (e.g. crop fertilization and deposition from combustion sources) is increasing so that N now threatens ecosystem sustainability around the world by causing terrestrial and aquatic eutrophication and acidification. It is important to better understand the capacity of AMF to directly uptake N from soils and transfer it to host plants because this process may increase N recycling and retention within ecosystems. In addition to understanding the role of AMF in the N cycle in the present day it is important to understand how AMF function may change as global change proceeds. Currently the net effects of N enrichment and elevated temperature predicted with global change on AMF are unknown. In this study, we examined the effects of N enrichment by simulated N‐deposition loading, elevated temperatures expected by future global changes and their interactions on growth and AMF‐mediated N acquisition of switchgrass (Panicum virgatum var. Alamo), an important species for biofuel production. Switchgrass plants were grown in microcosm units that divided mycorrhizal roots from AMF hyphae and organic residues enriched with ¹⁵N by compartments separated by an air gap to reduce N diffusion. While AMF did not enhance switchgrass biomass, mycorrhizas significantly increased ¹⁵N in shoots and total shoot N. Neither N enrichment nor elevated temperatures influenced this mycorrhizal‐mediated N uptake and transfer. Results from this study can aid in developing sustainable bioethanol and switchgrass production practices that are less reliant on synthetic fertilizers and more dependent on internal N recycling from AMF.     

Trophoblasts regulate the placental hematopoietic niche through PDGF-B signaling

The placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRβ, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.     

Ultrasound biomicroscopy for longitudinal studies of carotid plaque development in mice: validation with histological endpoints

Atherosclerosis is responsible for the death of thousands of Americans each year. The carotid constriction model of plaque development has recently been presented as a model for unstable plaque formation in mice. In this study we 1) validate ultrasound biomicroscopy (UBM) for the determination of carotid plaque size, percent stenosis, and plaque development in live animals, 2) determine the sensitivity of UBM in detecting changes in blood flow induced by carotid constriction and 3) test whether plaque formation can be predicted from blood flow parameters measured by UBM. Carotid plaques were induced by surgical constriction in Apo E−/− mice. Arteries were imaged bi-weekly by UBM, at which time PW-Doppler measurements of proximal blood flow, as well as plaque length and percent stenosis were determined. Histology was performed 9 weeks post surgery. When compared to whole mount post-mortem measurements, UBM accurately reported carotid plaque length. Percent stenosis, based on transverse B-mode UBM measurements, correlated well with that calculated from histological sections. PW-Doppler revealed that constriction reduced maximum systolic velocity (v_max) and duration of the systolic velocity peak (t_s/t_t). Pre-plaque (2 week post-surgery) PW-Doppler parameters (v_max and t_s/t_t) were correlated with plaque length at 9 weeks, and were predictive of plaque formation. Correlation of initiating PW-Doppler parameters (v_max and t_s/t_t) with resulting plaque length established the degree of flow disturbance required for subsequent plaque development and demonstrated its power for predicting plaque development.