Low-pathogenicity Mycoplasma spp. alter human monocyte and macrophage function and are highly prevalent among patients with ventilator-acquired pneumonia.

BACKGROUND: Ventilator-acquired pneumonia (VAP) remains a significant problem within intensive care units (ICUs). There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent among patients with VAP than previously recognised, and that these organisms potentially impair immune cell function. METHODS AND SETTING: 159 patients were recruited from 12 UK ICUs. All patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage (BAL). VAP was defined as growth of organisms at >10(4) colony forming units per ml of BAL fluid on conventional culture. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. 36 healthy donors underwent BAL for comparison. Additionally, healthy donor monocytes and macrophages were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed. RESULTS: Mycoplasmataceae were found in 49% (95% CI 33% to 65%) of patients with VAP, compared with 14% (95% CI 9% to 25%) of patients without VAP. Patients with sterile BAL fluid had a similar prevalence to healthy donor BAL fluid (10% (95% CI 4% to 20%) vs 8% (95% CI 2% to 22%)). The most common organism identified was M. salivarium. Blood monocytes from healthy volunteers incubated with M. salivarium displayed an impaired TNF-α response to lipopolysaccharide (p=0.0003), as did monocyte-derived macrophages (MDMs) (p=0.024). MDM exposed to M. salivarium demonstrated impaired phagocytosis (p=0.005). DISCUSSION AND CONCLUSIONS: This study demonstrates a high prevalence of Mycoplasmataceae among patients with VAP, with a markedly lower prevalence among patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The most common organism found, M. salivarium, is able to alter the functions of key immune cells. Mycoplasmataceae may contribute to VAP pathogenesis.This study was funded by the Hospital Infection Society, Wellcome Trust/Department of Health Health Innovation Challenge Fund (HICF)(0510/078) and Sir Jules Thorn Charitable Trust (03/JTA).This is the final version of the article. It first appeared from BMJ Publishing Group via http://dx.doi.org/10.1136/thoraxjnl-2015-20805

Diagnosing Alzheimer's Disease from Circulating Blood Leukocytes Using a Fluorescent Amyloid Probe

BACKGROUND: Toxic amyloid-β (Aβ) peptides aggregate into higher molecular weight assemblies and accumulate not only in the extracellular space, but also in the walls of blood vessels in the brain, increasing their permeability, and promoting immune cell migration and activation. Given the prominent role of the immune system, phagocytic blood cells may contact pathological brain materials. OBJECTIVE: To develop a novel method for early Alzheimer's disease (AD) detection, we used blood leukocytes, that could act as "sentinels" after trafficking through the brain microvasculature, to detect pathological amyloid by labelling with a conformationally-sensitive fluorescent amyloid probe and imaging with confocal spectral microscopy. METHODS: Formalin-fixed peripheral blood mononuclear cells (PBMCs) from cognitively healthy control (HC) subjects, mild cognitive impairment (MCI) and AD patients were stained with the fluorescent amyloid probe K114, and imaged. Results were validated against cerebrospinal fluid (CSF) biomarkers and clinical diagnosis. RESULTS: K114-labeled leukocytes exhibited distinctive fluorescent spectral signatures in MCI/AD subjects. Comparing subjects with single CSF biomarker-positive AD/MCI to negative controls, our technique yielded modest AUCs, which improved to the 0.90 range when only MCI subjects were included in order to measure performance in an early disease state. Combining CSF Aβ 42 and t-Tau metrics further improved the AUC to 0.93. CONCLUSION: Our method holds promise for sensitive detection of AD-related protein misfolding in circulating leukocytes, particularly in the early stages of disease

The Red Sea, Coastal Landscapes, and Hominin Dispersals

This chapter provides a critical assessment of environment, landscape and resources in the Red Sea region over the past five million years in relation to archaeological evidence of hominin settlement, and of current hypotheses about the role of the region as a pathway or obstacle to population dispersals between Africa and Asia and the possible significance of coastal colonization. The discussion assesses the impact of factors such as topography and the distribution of resources on land and on the seacoast, taking account of geographical variation and changes in geology, sea levels and palaeoclimate. The merits of northern and southern routes of movement at either end of the Red Sea are compared. All the evidence indicates that there has been no land connection at the southern end since the beginning of the Pliocene period, but that short sea crossings would have been possible at lowest sea-level stands with little or no technical aids. More important than the possibilities of crossing the southern channel is the nature of the resources available in the adjacent coastal zones. There were many climatic episodes wetter than today, and during these periods water draining from the Arabian escarpment provided productive conditions for large mammals and human populations in coastal regions and eastwards into the desert. During drier episodes the coastal region would have provided important refugia both in upland areas and on the emerged shelves exposed by lowered sea level, especially in the southern sector and on both sides of the Red Sea. Marine resources may have offered an added advantage in coastal areas, but evidence for their exploitation is very limited, and their role has been over-exaggerated in hypotheses of coastal colonization

Roaring high and low: composition and possible functions of the Iberian stag's vocal repertoire

We provide a detailed description of the rutting vocalisations of free-ranging male Iberian deer (Cervus elaphus hispanicus, Hilzheimer 1909), a geographically isolated and morphologically differentiated subspecies of red deer Cervus elaphus. We combine spectrographic examinations, spectral analyses and automated classifications to identify different call types, and compare the composition of the vocal repertoire with that of other red deer subspecies. Iberian stags give bouts of roars (and more rarely, short series of barks) that are typically composed of two different types of calls. Long Common Roars are mostly given at the beginning or at the end of the bout, and are characterised by a high fundamental frequency (F0) resulting in poorly defined formant frequencies but a relatively high amplitude. In contrast, Short Common Roars are typically given in the middle or at the end of the bout, and are characterised by a lower F0 resulting in relatively well defined vocal tract resonances, but low amplitude. While we did not identify entirely Harsh Roars (as described in the Scottish red deer subspecies (Cervus elaphus scoticus), a small percentage of Long Common Roars contained segments of deterministic chaos. We suggest that the evolution of two clearly distinct types of Common Roars may reflect divergent selection pressures favouring either vocal efficiency in high pitched roars or the communication of body size in low-pitched, high spectral density roars highlighting vocal tract resonances. The clear divergence of the Iberian red deer vocal repertoire from those of other documented European red deer populations reinforces the status of this geographical variant as a distinct subspecies

The Druze: A Population Genetic Refugium of the Near East

BACKGROUND: Phylogenetic mitochondrial DNA haplogroups are highly partitioned across global geographic regions. A unique exception is the X haplogroup, which has a widespread global distribution without major regions of distinct localization. PRINCIPAL FINDINGS: We have examined mitochondrial DNA sequence variation together with Y-chromosome-based haplogroup structure among the Druze, a religious minority with a unique socio-demographic history residing in the Near East. We observed a striking overall pattern of heterogeneous parental origins, consistent with Druze oral tradition, together with both a high frequency and a high diversity of the mitochondrial DNA (mtDNA) X haplogroup within a confined regional subpopulation. Furthermore demographic modeling indicated low migration rates with nearby populations. CONCLUSIONS: These findings were enabled through the use of a paternal kindred based sampling approach, and suggest that the Galilee Druze represent a population isolate, and that the combination of a high frequency and diversity of the mtDNA X haplogroup signifies a phylogenetic refugium, providing a sample snapshot of the genetic landscape of the Near East prior to the modern age

clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion

BACKGROUND: The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1. METHODOLOGY/PRINCIPAL FINDINGS: Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36. CONCLUSIONS/SIGNIFICANCE: Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications

The Use of Haplotypes in the Identification of Interaction between SNPs

Although haplotypes can provide great insight into the complex relationships between functional polymorphisms at a locus, their use in modern association studies has been limited. This is due to our inability to directly observe haplotypes in studies of unrelated individuals, but also to the extra complexity involved in their analysis and the difficulty in identifying which is the truly informative haplotype. Using a series of simulations, we tested a number of different models of a haplotype carrying two functional single nucleotide polymorphisms (SNPs) to assess the ability of haplotypic analysis to identify functional interactions between SNPs at the same locus. We found that, when phase is known, analysis of the haplotype is more powerful than analysis of the individual SNPs. The difference between the two approaches becomes less either as an increasing number of non-informative SNPs are included, or when the haplotypic phase is unknown, while in both cases the SNP association becomes progressively better at identifying the association. Our results suggest that when novel genotyping and bioinformatics methods are available to reconstruct haplotypic phase, this will permit the emergence of a new wave of haplotypic analysis able to consider interactions between SNPs with increased statistical power.</p

Inclusion of maintenance energy improves the intracellular flux predictions of CHO

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.COMET center acib: Next Generation Bioproduction, which is funded by BMK, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria and Vienna Business Agency in the framework of COMET - Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG; D.S., J.S., M.W., M.H., D. E.R. This work has also been supported by the PhD program BioToP of the Austrian Science Fund (FWF Project W1224)info:eu-repo/semantics/publishedVersio

Species Differentiation on a Dynamic Landscape: Shifts in Metapopulation Genetic Structure Using the Chronology of the Hawaiian Archipelago

Species formation during adaptive radiation often occurs in the context of a changing environment. The establishment and arrangement of populations, in space and time, sets up ecological and genetic processes that dictate the rate and pattern of differentiation. Here, we focus on how a dynamic habitat can affect genetic structure, and ultimately, differentiation among populations. We make use of the chronology and geographical history provided by the Hawaiian archipelago to examine the initial stages of population establishment and genetic divergence. We use data from a set of 6 spider lineages that differ in habitat affinities, some preferring low elevation habitats with a longer history of connection, others being more specialized for high elevation and/or wet forest, some with more general habitat affinities. We show that habitat preferences associated with lineages are important in ecological and genetic structuring. Lineages that have more restricted habitat preferences are subject to repeated episodes of isolation and fragmentation as a result of lava flows and vegetation succession. The initial dynamic set up by the landscape translates over time into discrete lineages. Further work is needed to understand how genetic changes interact with a changing set of ecological interactions amongst a shifting mosaic of landscapes to achieve species formation

Whole genome association mapping by incompatibilities and local perfect phylogenies

BACKGROUND: With current technology, vast amounts of data can be cheaply and efficiently produced in association studies, and to prevent data analysis to become the bottleneck of studies, fast and efficient analysis methods that scale to such data set sizes must be developed. RESULTS: We present a fast method for accurate localisation of disease causing variants in high density case-control association mapping experiments with large numbers of cases and controls. The method searches for significant clustering of case chromosomes in the "perfect" phylogenetic tree defined by the largest region around each marker that is compatible with a single phylogenetic tree. This perfect phylogenetic tree is treated as a decision tree for determining disease status, and scored by its accuracy as a decision tree. The rationale for this is that the perfect phylogeny near a disease affecting mutation should provide more information about the affected/unaffected classification than random trees. If regions of compatibility contain few markers, due to e.g. large marker spacing, the algorithm can allow the inclusion of incompatibility markers in order to enlarge the regions prior to estimating their phylogeny. Haplotype data and phased genotype data can be analysed. The power and efficiency of the method is investigated on 1) simulated genotype data under different models of disease determination 2) artificial data sets created from the HapMap ressource, and 3) data sets used for testing of other methods in order to compare with these. Our method has the same accuracy as single marker association (SMA) in the simplest case of a single disease causing mutation and a constant recombination rate. However, when it comes to more complex scenarios of mutation heterogeneity and more complex haplotype structure such as found in the HapMap data our method outperforms SMA as well as other fast, data mining approaches such as HapMiner and Haplotype Pattern Mining (HPM) despite being significantly faster. For unphased genotype data, an initial step of estimating the phase only slightly decreases the power of the method. The method was also found to accurately localise the known susceptibility variants in an empirical data set – the ΔF508 mutation for cystic fibrosis – where the susceptibility variant is already known – and to find significant signals for association between the CYP2D6 gene and poor drug metabolism, although for this dataset the highest association score is about 60 kb from the CYP2D6 gene. CONCLUSION: Our method has been implemented in the Blossoc (BLOck aSSOCiation) software. Using Blossoc, genome wide chip-based surveys of 3 million SNPs in 1000 cases and 1000 controls can be analysed in less than two CPU hours