A crucial requirement for Hedgehog signaling in small cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.     

Epidemiological tracking and population assignment of the non-clonal bacterium, Burkholderia pseudomallei

Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST). These populations correlate with the major foci of endemicity (Australia and Southeast Asia). Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs). Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic characteristics and provides a rapid reference for epidemiologists wishing to track the origin of infection without the need to compile population data and learn population assignment algorithms.     

Diagnostic accuracy of real-time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-control study

Background

Rapid PCR-based tests for the diagnosis of leptospirosis can provide information that contributes towards early patient management, but these have not been adopted in Thailand. Here, we compare the diagnostic sensitivity and specificity of two real-time PCR assays targeting rrs or lipL32 for the diagnosis of leptospirosis in northeast Thailand.

Methods/Principal Findings

A case-control study of 266 patients (133 cases of leptospirosis and 133 controls) was constructed to evaluate the diagnostic sensitivity and specificity (DSe & DSp) of both PCR assays. The median duration of illness prior to admission of cases was 4 days (IQR 2–5 days; range 1–12 days). DSe and DSp were determined using positive culture and/or microscopic agglutination test (MAT) as the gold standard. The DSe was higher for the rrs assay than the lipL32 assay (56%, (95% CI 47–64%) versus 43%, (95% CI 34–52%), p<0.001). No cases were positive for the lipL32 assay alone. There was borderline evidence to suggest that the DSp of the rrs assay was lower than the lipL32 assay (90% (95% CI 83–94%) versus 93%, (95%CI 88–97%), p = 0.06). Nine controls gave positive reactions for both assays and 5 controls gave a positive reaction for the rrs assay alone. The DSe of the rrs and lipL32 assays were high in the subgroup of 39 patients who were culture positive for Leptospira spp. (95% and 87%, respectively, p = 0.25).

Conclusions/Significance

Early detection of Leptospira using PCR is possible for more than half of patients presenting with leptospirosis and could contribute to individual patient care.     

Diversity of the microeukaryotic community in sulfide-rich Zodletone Spring (Oklahoma)

The microeukaryotic community in Zodletone Spring, a predominantly anaerobic sulfide and sulfur-rich spring, was examined using an 18S rRNA gene cloning and sequencing approach. The majority of the 288 clones sequenced from three different locations at Zodletone Spring belonged to the Stramenopiles, Alveolata, and Fungi, with members of the phylum Cercozoa, order Diplomonadida, and family Jakobidae representing a minor fraction of the clone library. No sequences suggesting the presence of novel kingdom level diversity were detected in any of the three libraries. A large fraction of stramenopile clones encountered were monophyletic with either members of the genus Cafeteria (order Bicosoecida) or members of the order Labyrinthulida (slime nets), both of which have so far been encountered mainly in marine habitats. The majority of the observed fungal clone sequences belonged to the ascomycetous yeasts (order Saccharomycetales), were closely related to yeast genera within the Hymenobasidiomycetes (phylum Basidiomycetes), or formed a novel fungal lineage with several previously published or database-deposited clones. To determine whether the unexpected abundance of fungal sequences in Zodletone Spring clone libraries represents a general pattern in anaerobic habitats, we generated three clone libraries from three different anaerobic settings (anaerobic sewage digester, pond sediment, and hydrocarbon-exposed aquifer sediments) and partially sequenced 210 of these clones. Phylogenetic analysis indicated that clone sequences belonging to the kingdom Fungi represent a significant fraction of all three clone libraries, an observation confirmed by phospholipid fatty acid and ergosterol analysis. Overall, this work reveals an unexpected abundance of Fungi in anaerobic habitats, describes a novel, yet-uncultured group of Fungi that appears to be widespread in anaerobic habitats, and indicates that several of the previously considered marine protists could also occur in nonmarine habitats.     

Gender differences in human immunodeficiency virus type 1-specific CD8 responses in the reproductive tract and colon following nasal peptide priming and modified vaccinia virus Ankara boosting

Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.     

Siderophore uptake in bacteria and the battle for iron with the host; a bird’s eye view

Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe³⁺). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.     

Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes

Staphylococcus aureus is the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting, S. aureus is a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and the S. aureus population structure, we compared 61 community-acquired invasive isolates of S. aureus and 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the seven S. aureus sequencing projects. The core genes common to all strains were identified, and 10 dominant lineages of S. aureus were clearly discriminated. Each lineage carried a unique combination of hundreds of "core variable" (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that the S. aureus gene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors.     

Effectiveness of a simplified method for isolation of Burkholderia pseudomallei from soil

Detection of environmental Burkholderia pseudomallei indicates a risk for melioidosis and is important for the development of a global risk map. We describe a simple method for detecting B. pseudomallei using direct culture of soil in enrichment broth. This gives a rate of positivity comparable to that obtained with a standard method but is cheaper and labor saving.     

Distribution of microbial biomass and potential for anaerobic respiration in Hanford Site 300 Area subsurface sediment

Subsurface sediments were recovered from a 52-m-deep borehole cored in the 300 Area of the Hanford Site in southeastern Washington State to assess the potential for biogeochemical transformation of radionuclide contaminants. Microbial analyses were made on 17 sediment samples traversing multiple geological units: the oxic coarse-grained Hanford formation (9 to 17.4 m), the oxic fine-grained upper Ringold formation (17.7 to 18.1 m), and the reduced Ringold formation (18.3 to 52 m). Microbial biomass (measured as phospholipid fatty acids) ranged from 7 to 974 pmols per g in discrete samples, with the highest numbers found in the Hanford formation. On average, strata below 17.4 m had 13-fold less biomass than those from shallower strata. The nosZ gene that encodes nitrous oxide reductase (measured by quantitative real-time PCR) had an abundance of 5 to 17 relative to that of total 16S rRNA genes below 18.3 m and <5 above 18.1 m. Most nosZ sequences were affiliated with Ochrobactrum anthropi (97 sequence similarity) or had a nearest neighbor of Achromobacter xylosoxidans (90 similarity). Passive multilevel sampling of groundwater geochemistry demonstrated a redox gradient in the 1.5-m region between the Hanford-Ringold formation contact and the Ringold oxic-anoxic interface. Within this zone, copies of the dsrA gene and Geobacteraceae had the highest relative abundance. The majority of dsrA genes detected near the interface were related to Desulfotomaculum spp. These analyses indicate that the region just below the contact between the Hanford and Ringold formations is a zone of active biogeochemical redox cycling.     

Diversity of Xenorhabdus and Photorhabdus spp. and Their Symbiotic Entomopathogenic Nematodes from Thailand

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand.