Determination of bacterial quantity by sonication in vacum-assisted closure (VAC) foams used for treatment of chronic wounds

Background: Negative pressure wound treatment is increasingly used through a Vacuum-Assisted Closure (VAC) device in complex wound situations. For this purpose, sterile polyurethane (PU) and polyvinyl alcohol (PVA) foam dressings are fitted to the wound size and covered with an adhesive drape to create an airtight seal. Little information exists about the type and quantity of microorganisms within the foams. Therefore, we investigated VAC foams after removal from the wound using a validated method (sonication) to detect the bacterial bioburden in the foam consisting as microbial biofilms.Methods: We prospectively included VAC foams (PU and PVA, KCI, Rümlamg, Switzerland) without antibacterial additions (e.g. silver), which were removed from wounds in patients with chronic ulcers from January 2007 through December 2008. Excluded were patients with acute wound infection, necrotizing fasciitis, underlying osteomyelitis or implant. Removed foams from regular changes of dressing were aseptically placed in a container with 100 ml sterile Ringer's solution. Within 4 hours after removal, foams were sonicated for 5 min at 40 kHz (as described in NEJM 2007;357:654). The resulting sonication fluid was cultured at 37°C on aerobic blood agar plates for 5 days. Microbes were quantified as No. of colony-forming units (CFU)/ml sonication fluid and identified to the species level.Results: A total of 68 foams (38 PU and 30 PVA) from 55 patients were included in the study (median age 71 years; range 33-88 years, 57% were man). Foams were removed from the following anatomic sites: sacrum (n=29), ischium (n=18), heel (n=13), calves (n=6) and ankle (n=2). The median duration of being in place was 3 days (range, 1-8 days). In all 68 foams, bacteria were found in large quantities (median 105 CFU/ml, range 102-7 CFU/ml sonication fluid. No differences were found between PU and PVA foams. One type of organisms was found in 11 (16%), two in 17 (24%) and 3 or more in 40 (60%) foams. Gram-negative rods (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa) were isolated in 70%, followed by Staphylococcus aureus (20%), koagulase-negative staphylococci, streptococci (8%), and enterococci (2%).Conclusion: With sonication, a high density of bacteria present in VAC foams was demonstrated after a median of 3 days. Future studies are needed to investigate whether antimicrobial-impregnated foams can reduce the bacterial load in foams and potentially improve wound healing.

Use of flow cytometric methods for single-cell analysis in environmental microbiology

Flow cytometry (FCM) is emerging as an important tool in environmental microbiology. Although flow cytometry applications have to date largely been restricted to certain specialized fields of microbiology, such as the bacterial cell cycle and marine phytoplankton communities, technical advances in instrumentation and methodology are leading to its increased popularity and extending its range of applications. Here we will focus on a number of recent flow cytometry developments important for addressing questions in environmental microbiology. These include (i) the study of microbial physiology under environmentally relevant conditions, (ii) new methods to identify active microbial populations and to isolate previously uncultured microorganisms, and (iii) the development of high-throughput autofluorescence bioreporter assays

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Genetic diversity in widespread species is not congruent with species richness in alpine plant communities.

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

Improving generalized regression analysis for the spatial prediction of forest communities

Aim This study used data from temperate forest communities to assess: (1) five different stepwise selection methods with generalized additive models, (2) the effect of weighting absences to ensure a prevalence of 0.5, (3) the effect of limiting absences beyond the environmental envelope defined by presences, (4) four different methods for incorporating spatial autocorrelation, and (5) the effect of integrating an interaction factor defined by a regression tree on the residuals of an initial environmental model. Location State of Vaud, western Switzerland. Methods Generalized additive models (GAMs) were fitted using the grasp package (generalized regression analysis and spatial predictions, http://www.cscf.ch/grasp). Results Model selection based on cross-validation appeared to be the best compromise between model stability and performance (parsimony) among the five methods tested. Weighting absences returned models that perform better than models fitted with the original sample prevalence. This appeared to be mainly due to the impact of very low prevalence values on evaluation statistics. Removing zeroes beyond the range of presences on main environmental gradients changed the set of selected predictors, and potentially their response curve shape. Moreover, removing zeroes slightly improved model performance and stability when compared with the baseline model on the same data set. Incorporating a spatial trend predictor improved model performance and stability significantly. Even better models were obtained when including local spatial autocorrelation. A novel approach to include interactions proved to be an efficient way to account for interactions between all predictors at once. Main conclusions Models and spatial predictions of 18 forest communities were significantly improved by using either: (1) cross-validation as a model selection method, (2) weighted absences, (3) limited absences, (4) predictors accounting for spatial autocorrelation, or (5) a factor variable accounting for interactions between all predictors. The final choice of model strategy should depend on the nature of the available data and the specific study aims. Statistical evaluation is useful in searching for the best modelling practice. However, one should not neglect to consider the shapes and interpretability of response curves, as well as the resulting spatial predictions in the final assessment.

Bacterial transcriptional regulators for degradation pathways of aromatic compounds.

Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways.

Protection from lethal gram-negative bacterial sepsis by targeting Toll-like receptor 4.

Toll-like receptor 4 (TLR4), the signal-transducing molecule of the LPS receptor complex, plays a fundamental role in the sensing of LPS from gram-negative bacteria. Activation of TLR4 signaling pathways by LPS is a critical upstream event in the pathogenesis of gram-negative sepsis, making TLR4 an attractive target for novel antisepsis therapy. To validate the concept of TLR4-targeted treatment strategies in gram-negative sepsis, we first showed that TLR4(-/-) and myeloid differentiation primary response gene 88 (MyD88)(-/-) mice were fully resistant to Escherichia coli-induced septic shock, whereas TLR2(-/-) and wild-type mice rapidly died of fulminant sepsis. Neutralizing anti-TLR4 antibodies were then generated using a soluble chimeric fusion protein composed of the N-terminal domain of mouse TLR4 (amino acids 1-334) and the Fc portion of human IgG1. Anti-TLR4 antibodies inhibited intracellular signaling, markedly reduced cytokine production, and protected mice from lethal endotoxic shock and E. coli sepsis when administered in a prophylactic and therapeutic manner up to 13 h after the onset of bacterial sepsis. These experimental data provide strong support for the concept of TLR4-targeted therapy for gram-negative sepsis.

On landslide risk, resilience and vulnerability of mountain communities in Central-Eastern Nepal

Executive SummaryIn Nepal, landslides are one of the major natural hazards after epidemics, killing over 100 persons per year. However, this figure is an underreported reflection of the actual impact that landslides have on livelihoods and food security in rural Nepal. With predictions of more intense rainfall patterns, landslide occurrence in the Himalayas is likely to increase and continue to be one of the major impediments to development. Due to the remoteness of many localities and lack of resources, responsibilities for disaster preparedness and response in mountain areas usually lie with the communities themselves. Everyday life is full of risk in mountains of Nepal. This is why mountain populations, as well as other populations living in harsh conditions have developed a number of coping strategies for dealing with adverse situations. Perhaps due to the dispersed and remote nature of landslides in Nepal, there have been few studies on vulnerability, coping- and mitigation strategies of landslide affected populations. There are also few recommendations available to guide authorities and populations how to reduce losses due to landslides in Nepal, and even less so, how to operationalize resilience and vulnerability.Many policy makers, international donors, NGOs and national authorities are currently asking what investments are needed to increase the so-called 'resilience' of mountain populations to deal with climate risks. However, mountain populations are already quite resilient to seasonal fluctuations, temperature variations, rainfall patterns and market prices. In spite of their resilience, they continue to live in places at risk due to high vulnerability caused by structural inequalities: access to land, resources, markets, education. This interdisciplinary thesis examines the concept of resilience by questioning its usefulness and validity as the current goal of international development and disaster risk reduction policies, its conceptual limitations and its possible scope of action. The goal of this study is two-fold: to better define and distinguish factors and relationships between resilience, vulnerability, capacities and risk; and to test and improve a participatory methodology for evaluating landslide risk that can serve as a guidance tool for improving community-based disaster risk reduction. The objective is to develop a simple methodology that can be used by NGOs, local authorities and communities to reduce losses from landslides.Through its six case studies in Central-Eastern Nepal, this study explores the relation between resilience, vulnerability and landslide risk based on interdisciplinary methods, including geological assessments of landslides, semi-structured interviews, focus groups and participatory risk mapping. For comparison, the study sites were chosen in Tehrathum, Sunsari and Dolakha Districts of Central/Eastern Nepal, to reflect a variety of landslide types, from chronic to acute, and a variety of communities, from very marginalized to very high status. The study uses the Sustainable Livelihoods Approach as its conceptual basis, which is based on the notion that access and rights to resources (natural, human/institutional, economic, environmental, physical) are the basis for coping with adversity, such as landslides. The study is also intended as a contribution to the growing literature and practices on Community Based Disaster Risk Reduction specifically adapted to landslide- prone areas.In addition to the six case studies, results include an indicator based methodology for assessing and measuring vulnerability and resilience, a composite risk assessment methodology, a typology of coping strategies and risk perceptions and a thorough analysis of the relation between risk, vulnerability and resilience. The methodology forassessing vulnerability, resilience and risk is relatively cost-effective and replicable in a low-data environment. Perhaps the major finding is that resilience is a process that defines a community's (or system's) capacity to rebound following adversity but it does not necessarily reduce vulnerability or risk, which requires addressing more structural issues related to poverty. Therefore, conclusions include a critical view of resilience as a main goal of international development and disaster risk reduction policies. It is a useful concept in the context of recovery after a disaster but it needs to be addressed in parallel with vulnerability and risk.This research was funded by an interdisciplinary grant (#26083591) from the Swiss National Science Foundation for the period 2009-2011 and a seed grant from the Faculty of Geosciences and Environment at the University of Lausanne in 2008.Résumé en françaisAu Népal, les glissements de terrain sont un des aléas les plus dévastateurs après les épidémies, causant 100 morts par an. Pourtant, ce chiffre est une sous-estimation de l'impact réel de l'effet des glissements sur les moyens de subsistance et la sécurité alimentaire au Népal. Avec des prévisions de pluies plus intenses, l'occurrence des glissements dans les Himalayas augmente et présente un obstacle au développement. Du fait de l'éloignement et du manque de ressources dans les montagnes au Népal, la responsabilité de la préparation et la réponse aux catastrophes se trouve chez les communautés elles-mêmes. Le risque fait partie de la vie quotidienne dans les montagnes du Népal. C'est pourquoi les populations montagnardes, comme d'autres populations vivant dans des milieux contraignants, ont développé des stratégies pour faire face aux situations défavorables. Peu d'études existent sur la vulnérabilité, ceci étant probablement dû à l'éloignement et pourtant, les stratégies d'adaptation et de mitigation des populations touchées par des glissements au Népal existent.Beaucoup de décideurs politiques, bailleurs de fonds, ONG et autorités nationales se demandent quels investissements sont nécessaires afin d'augmenter la 'resilience' des populations de montagne pour faire face aux changements climatiques. Pourtant, ces populations sont déjà résilientes aux fluctuations des saisons, des variations de température, des pluies et des prix des marchés. En dépit de leur résilience, ils continuent de vivre dans des endroits à fort risque à cause des vulnérabilités créées par les inégalités structurelles : l'accès à la terre, aux ressources, aux marchés et à l'éducation. Cette thèse interdisciplinaire examine le concept de la résilience en mettant en cause son utilité et sa validité en tant que but actuel des politiques internationales de développement et de réduction des risques, ainsi que ses limitations conceptuelles et ses possibles champs d'action. Le but de cette étude est double : mieux définir et distinguer les facteurs et relations entre la résilience, la vulnérabilité, les capacités et le risque ; Et tester et améliorer une méthode participative pour évaluer le risque des glissements qui peut servir en tant qu'outil indicatif pour améliorer la réduction des risques des communautés. Le but est de développer une méthodologie simple qui peut être utilisée par des ONG, autorités locales et communautés pour réduire les pertes dues aux glissements.A travers les études de cas au centre-est du Népal, cette étude explore le rapport entre la résilience, la vulnérabilité et les glissements basée sur des méthodes interdisciplinaires ; Y sont inclus des évaluations géologiques des glissements, des entretiens semi-dirigés, des discussions de groupes et des cartes de risques participatives. Pour la comparaison, les zones d'études ont été sélectionnées dans les districts de Tehrathum, Sunsari et Dolakha dans le centre-est du Népal, afin de refléter différents types de glissements, de chroniques à urgents, ainsi que différentes communautés, variant de très marginalisées à très haut statut. Pour son cadre conceptuel, cette étude s'appuie sur l'approche de moyens de subsistance durable, qui est basée sur les notions d'accès et de droit aux ressources (naturelles, humaines/institutionnelles, économiques, environnementales, physiques) et qui sont le minimum pour faire face à des situations difficiles, comme des glissements. Cette étude se veut aussi une contribution à la littérature et aux pratiques en croissantes sur la réduction des risques communautaires, spécifiquement adaptées aux zones affectées par des glissements.En plus des six études de cas, les résultats incluent une méthodologie basée sur des indicateurs pour évaluer et mesurer la vulnérabilité et la résilience, une méthodologie sur le risque composé, une typologie de stratégies d'adaptation et perceptions des risques ainsi qu'une analyse fondamentale de la relation entre risque, vulnérabilité et résilience. Les méthodologies pour l'évaluation de la vulnérabilité, de la résilience et du risque sont relativement peu coûteuses et reproductibles dans des endroits avec peu de données disponibles. Le résultat probablement le plus pertinent est que la résilience est un processus qui définit la capacité d'une communauté (ou d'un système) à rebondir suite à une situation défavorable, mais qui ne réduit pas forcement la vulnérabilité ou le risque, et qui requiert une approche plus fondamentale s'adressant aux questions de pauvreté. Les conclusions incluent une vue critique de la résilience comme but principal des politiques internationales de développement et de réduction des risques. C'est un concept utile dans le contexte de la récupération après une catastrophe mais il doit être pris en compte au même titre que la vulnérabilité et le risque.Cette recherche a été financée par un fonds interdisciplinaire (#26083591) du Fonds National Suisse pour la période 2009-2011 et un fonds de préparation de recherches par la Faculté des Géosciences et Environnement à l'Université de Lausanne en 2008.

Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation.

Although Drosophila systemic immunity is extensively studied, little is known about the fly's intestine-specific responses to bacterial infection. Global gene expression analysis of Drosophila intestinal tissue to oral infection with the Gram-negative bacterium Erwinia carotovora revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. Ingestion of bacteria had a dramatic impact on the physiology of the gut that included modulation of stress response and increased stem cell proliferation and epithelial renewal. Our data suggest that gut homeostasis is maintained through a balance between cell damage due to the collateral effects of bacteria killing and epithelial repair by stem cell division. The Drosophila gut provides a powerful model to study the integration of stress and immunity with pathways associated with stem cell control, and this study should prove to be a useful resource for such further studies.

Correlation between placental bacterial culture results and histological chorioamnionitis: a prospective study on 376 placentas.

OBJECTIVE: To study the correlation between the bacteriological and histopathological findings in placentas from women with suspected or proven chorioamnionitis (CA). METHODS: Over a 1-year period, 376 placentas were prospectively collected and processed for bacteriological and pathological studies in cases of confirmed or suspected maternal or neonatal infection. RESULTS: Histological CA was diagnosed in 26.9% of placentas (101/376), and 27.7% (28/101) of these placentas had positive bacteriological cultures. A monomicrobial culture, mainly represented by Gram-positive cocci and Gram-negative bacilli, was identified in 27% of the positive bacterial cultures. The proportion of positive cultures was higher (p=0.03) when CA was associated with funisitis, as compared with placental samples with early CA. In placentas without histological CA, bacteriological cultures were mostly negative (230/275), although pathogenic bacteria were identified in 16.3% of them (45/275). CONCLUSIONS: The histological and bacteriological results were concordant in about 70% of the examined placentas, with 61.1% negative cases (CA absent and negative bacterial cultures), and only 7.4% placentas with positive histological and bacteriological results. Discordant results (positive histology with negative bacteriology) were obtained in placentas with early CA documented by histology although possibly in relation with antibiotic prophylaxis and the presence of fastidious bacteria. Conversely, negative histology with positive bacteriology could be explained by the presence of an early-stage bacterial infection that has not yet led to detectable microscopic lesions.

The role of phylogeny in the spatial distributions and assembly of mountain plant communities.

A major challenge in community ecology is a thorough understanding of the processes that govern the assembly and composition of communities in time and space. The growing threat of climate change to the vascular plant biodiversity of fragile ecosystems such as mountains has made it equally imperative to develop comprehensive methodologies to provide insights into how communities are assembled. In this perspective, the primary objective of this PhD thesis is to contribute to the theoretical and methodological development of community ecology, by proposing new solutions to better detect the ecological and evolutionary processes that govern community assembly. As phylogenetic trees provide by far, the most advanced tools to integrate the spatial, ecological and evolutionary dynamics of plant communities, they represent the cornerstone on which this work was based. In this thesis, I proposed new solutions to: (i) reveal trends in community assembly on phylogenies, depicted by the transition of signals at the nodes of the different species and lineages responsible for community assembly, (ii) contribute to evidence the importance of evolutionarily labile traits in the distribution of mountain plant species. More precisely, I demonstrated that phylogenetic and functional compositional turnover in plant communities was driven by climate and human land use gradients mostly influenced by evolutionarily labile traits, (iii) predict and spatially project the phylogenetic structure of communities using species distribution models, to identify the potential distribution of phylogenetic diversity, as well as areas of high evolutionary potential along elevation. The altitudinal setting of the Diablerets mountains (Switzerland) provided an appropriate model for this study. The elevation gradient served as a compression of large latitudinal variations similar to a collection of islands within a single area, and allowed investigations on a large number of plant communities. Overall, this thesis highlights that stochastic and deterministic environmental filtering processes mainly influence the phylogenetic structure of plant communities in mountainous areas. Negative density-dependent processes implied through patterns of phylogenetic overdispersion were only detected at the local scale, whereas environmental filtering implied through phylogenetic clustering was observed at both the regional and local scale. Finally, the integration of indices of phylogenetic community ecology with species distribution models revealed the prospects of providing novel and insightful explanations on the potential distribution of phylogenetic biodiversity in high mountain areas. These results generally demonstrate the usefulness of phylogenies in inferring assembly processes, and are worth considering in the theoretical and methodological development of tools to better understand phylogenetic community structure.

Complete Nucleotide Sequence of Mouse Mammary Tumor Virus from JYG Chinese Wild Mice: Absence of Bacterial Insertion Sequences in the Cloned Viral gag Gene.

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.

Mesenteric fat-control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

Improved statistical analysis of low abundance phenomena in bimodal bacterial populations.

Accurate detection of subpopulation size determinations in bimodal populations remains problematic yet it represents a powerful way by which cellular heterogeneity under different environmental conditions can be compared. So far, most studies have relied on qualitative descriptions of population distribution patterns, on population-independent descriptors, or on arbitrary placement of thresholds distinguishing biological ON from OFF states. We found that all these methods fall short of accurately describing small population sizes in bimodal populations. Here we propose a simple, statistics-based method for the analysis of small subpopulation sizes for use in the free software environment R and test this method on real as well as simulated data. Four so-called population splitting methods were designed with different algorithms that can estimate subpopulation sizes from bimodal populations. All four methods proved more precise than previously used methods when analyzing subpopulation sizes of transfer competent cells arising in populations of the bacterium Pseudomonas knackmussii B13. The methods' resolving powers were further explored by bootstrapping and simulations. Two of the methods were not severely limited by the proportions of subpopulations they could estimate correctly, but the two others only allowed accurate subpopulation quantification when this amounted to less than 25% of the total population. In contrast, only one method was still sufficiently accurate with subpopulations smaller than 1% of the total population. This study proposes a number of rational approximations to quantifying small subpopulations and offers an easy-to-use protocol for their implementation in the open source statistical software environment R.

Bacterial and viral superantigens: roles in autoimmunity?

Superantigens are bacterial, viral, or retroviral proteins which can activate specifically a large proportion of T cells. In contrast with classical peptide antigen recognition, superantigens do not require processing to small peptides but act as complete or partially processed proteins. They can bind to major histocompatibility complex class II molecules and stimulate T cells expressing particular T cell receptor V beta chains. The other polymorphic parts of the T cell receptor, which are crucial for classical antigen recognition, are not important for this interaction. When this strategy is used a large proportion of the host immune system can be activated shortly after infection. The activated cells have a wide variety of antigen specificities. The ability to stimulate polyclonal B (IgG) as well as T cell responses raises possibilities of a role for superantigens in the induction of autoimmune diseases. Superantigens have been a great tool in the hands of immunologists in unravelling some of the basic mechanisms of tolerance and immunity.