PLoS ONE Alerts: New Articles

Egg Eviction Imposes a Recoverable Cost of Virulence in Chicks of a Brood Parasite

2009-11-11T08:00:00Z

Background

Chicks of virulent brood parasitic birds eliminate their nestmates and avoid costly competition for foster parental care. Yet, efforts to evict nest contents by the blind and naked common cuckoo Cuculus canorus hatchling are counterintuitive as both adult parasites and large older cuckoo chicks appear to be better suited to tossing the eggs and young of the foster parents.

Methodology/Principal Findings

Here we show experimentally that egg tossing imposed a recoverable growth cost of mass gain in common cuckoo chicks during the nestling period in nests of great reed warbler Acrocephalus arundinaceus hosts. Growth rates of skeletal traits and morphological variables involved in the solicitation of foster parental care remained similar between evictor and non-evictor chicks throughout development. We also detected no increase in predation rates for evicting nests, suggesting that egg tossing behavior by common cuckoo hatchlings does not increase the conspicuousness of nests.

Conclusion

The temporary growth cost of egg eviction by common cuckoo hatchlings is the result of constraints imposed by rejecter host adults and competitive nestmates on the timing and mechanism of parasite virulence.

New Policies, New Technologies: Modelling the Potential for Improved Smear Microscopy Services in Malawi

2009-11-10T08:00:00Z

Background

To quantify the likely impact of recent WHO policy recommendations regarding smear microscopy and the introduction of appropriate low-cost fluorescence microscopy on a) case detection and b) laboratory workload.

Methodology/Principal Findings

An audit of the laboratory register in an urban hospital, Lilongwe, Malawi, and the application of a simple modelling framework. The adoption of the new definition of a smear-positive case could directly increase case detection by up to 28%. Examining Ziehl-Neelsen (ZN) sputum smears for up to 10 minutes before declaring them negative has previously been shown to increase case detection (over and above that gained by the adoption of the new case definition) by 70% compared with examination times in routine practice. Three times the number of staff would be required to adequately examine the current workload of smears using ZN microscopy. Through implementing new policy recommendations and LED-based fluorescence microscopy the current laboratory staff complement could investigate the same number of patients, examining auramine-stained smears to an extent that is equivalent to a 10 minutes ZN smear examination.

Conclusions/Significance

Combined implementation of the new WHO recommendations on smear microscopy and LED-based fluorescence microscopy could result in substantial increases in smear positive case-detection using existing human resources and minimal additional equipment.

Inverse Symmetry in Complete Genomes and Whole-Genome Inverse Duplication

2009-11-09T08:00:00Z

The cause of symmetry is usually subtle, and its study often leads to a deeper understanding of the bearer of the symmetry. To gain insight into the dynamics driving the growth and evolution of genomes, we conducted a comprehensive study of textual symmetries in 786 complete chromosomes. We focused on symmetry based on our belief that, in spite of their extreme diversity, genomes must share common dynamical principles and mechanisms that drive their growth and evolution, and that the most robust footprints of such dynamics are symmetry related. We found that while complement and reverse symmetries are essentially absent in genomic sequences, inverse–complement plus reverse–symmetry is prevalent in complex patterns in most chromosomes, a vast majority of which have near maximum global inverse symmetry. We also discovered relations that can quantitatively account for the long observed but unexplained phenomenon of -mer skews in genomes. Our results suggest segmental and whole-genome inverse duplications are important mechanisms in genome growth and evolution, probably because they are efficient means by which the genome can exploit its double-stranded structure to enrich its code-inventory.

Determinants of the Relationship between Cytokine Production in Pregnant Women and Their Infants

2009-11-09T08:00:00Z

Exposure to environmental factors during fetal life and infancy is thought to play an important role in the early development of innate and adaptive immunity. The immunological relationship between mother and infant and the effect that environmental exposures have during pregnancy and early childhood have not been studied extensively. Here the production of cytokines was measured in 146 pairs of mothers and their 2- month-old infants. The effect of place of residence, socio-economic variables, parasitic infections as well as maternal and child characteristics on measured cytokine production was determined. Mothers producing high levels of IL-10, IFN-γ and IL-5 were more likely to have infants who also produced high levels of these cytokines either spontaneously (OR 2.6(95%CI 1.2–5.4), OR 2.9(CI 1.3–6.6), OR 11.2(CI 4.6–27.2), respectively) or in response to PHA (IL-10: OR 3.0(CI 1.4–6.6), IFN-γ: OR 2.0(CI 1.0–4.2), respectively) even after adjustment for potential confounding variables. This was not the case for TNF-α. In response to LPS, place of residence was a strong determinant of infant IL-10 (OR 0.2(CI 0.1–0.9)) and TNF-α (OR 0.3(CI 0.1–0.9)) production. Maternal protozoan infections was independently associated with reduced infant IL10 in response to PHA and to LPS as well as reduced TNF-α and IFN-γ in response to PHA. These results indicate strong relationship between maternal and infant's cellular immune responses even after taking into account many environmental influences that could affect infant's response directly or indirectly through uterine microenvironment. However, place of residence and intestinal infections may still directly affect the immune responses of the infant. Taken together, the study provides evidence for imprinted cytokine responses of an infant which may have implications for their reaction to incoming antigens, warranting further investigation into the role that genetics or epigenetics play in shaping the cytokine response by an infant to self or external antigens.

Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

2009-11-09T08:00:00Z

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ~30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ~138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

Phospholipase C-β4 Is Essential for the Progression of the Normal Sleep Sequence and Ultradian Body Temperature Rhythms in Mice

2009-11-09T08:00:00Z

Background

The sleep sequence: i) non-REM sleep, ii) REM sleep, and iii) wakefulness, is stable and widely preserved in mammals, but the underlying mechanisms are unknown. It has been shown that this sequence is disrupted by sudden REM sleep onset during active wakefulness (i.e., narcolepsy) in orexin-deficient mutant animals. Phospholipase C (PLC) mediates the signaling of numerous metabotropic receptors, including orexin receptors. Among the several PLC subtypes, the β4 subtype is uniquely localized in the geniculate nucleus of thalamus which is hypothesized to have a critical role in the transition and maintenance of sleep stages. In fact, we have reported irregular theta wave frequency during REM sleep in PLC-β4-deficient mutant (PLC-β4−/−) mice. Daily behavioral phenotypes and metabotropic receptors involved have not been analyzed in detail in PLC-β4−/− mice, however.

Methodology/Principal Findings

Therefore, we analyzed 24-h sleep electroencephalogram in PLC-β4−/− mice. PLC-β4−/− mice exhibited normal non-REM sleep both during the day and nighttime. PLC-β4−/− mice, however, exhibited increased REM sleep during the night, their active period. Also, their sleep was fragmented with unusual wake-to-REM sleep transitions, both during the day and nighttime. In addition, PLC-β4−/− mice reduced ultradian body temperature rhythms and elevated body temperatures during the daytime, but had normal homeothermal response to acute shifts in ambient temperatures (22°C–4°C). Within the most likely brain areas to produce these behavioral phenotypes, we found that, not orexin, but group-1 metabotropic glutamate receptor (mGluR)-mediated Ca²⁺ mobilization was significantly reduced in the dorsal lateral geniculate nucleus (LGNd) of PLC-β4−/− mice. Voltage clamp recordings revealed that group-1 mGluR-mediated currents in LGNd relay neurons (inward in wild-type mice) were outward in PLC-β4−/− mice.

Conclusions/Significance

These lines of evidence indicate that impaired LGNd relay, possibly mediated via group-1 mGluR, may underlie irregular sleep sequences and ultradian body temperature rhythms in PLC-β4−/− mice.

The Prevalence of Depression in White-European and South-Asian People with Impaired Glucose Regulation and Screen-Detected Type 2 Diabetes Mellitus

2009-11-09T08:00:00Z

Background

There is a clear relationship between depression and diabetes. However, the directionality of the relationship remains unclear and very little research has considered a multi-ethnic population. The aim of this study was to determine the prevalence of depression in a White-European (WE) and South-Asian (SA) population attending a community diabetes screening programme, and to explore the association of depression with screen-detected Type 2 diabetes mellitus (T2DM) and impaired glucose regulation (IGR).

Methodology/Principal Findings

Participants were recruited from general practices in Leicestershire (United Kingdom) between August 2004 and December 2007. 4682 WE (40–75 years) and 1327 SA participants (25–75 years) underwent an Oral Glucose Tolerance Test, detailed history, anthropometric measurements and completed the World Health Organisation-Five (WHO-5) Wellbeing Index. Depression was defined by a WHO-5 wellbeing score ≤13. Unadjusted prevalence of depression for people in the total sample with T2DM and IGR was 21.3% (21.6% in WE, 20.6% in SA, p = 0.75) and 26.0% (25.3% in WE, 28.9% in SA, p = 0.65) respectively. For people with normal glucose tolerance, the prevalence was 25.1% (24.9% in WE, 26.4% in SA, p = 0.86). Age-adjusted prevalences were higher for females than males. Odds ratios adjusted for age, gender, and ethnicity, showed no significant increase in prevalent depression for people with T2DM (OR = 0.95, 95%CI 0.62 to 1.45) or IGR (OR = 1.17, 95%CI 0.96 to1.42).

Conclusions

Prior to the knowledge of diagnosis, depression was not significantly more prevalent in people with screen detected T2DM or IGR. Differences in prevalent depression between WE and SA people were also not identified. In this multi-ethnic population, female gender was significantly associated with depression.

Rat Merkel Cells Are Mechanoreceptors and Osmoreceptors

2009-11-09T08:00:00Z

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca²⁺ channels and hypotonic-induced membrane deformation is known to lead to Ca²⁺ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification. Their properties were investigated through electrophysiological studies. Voltage-gated K⁺, Ca²⁺ and Ca²⁺-activated K⁺ (K_Ca) channels were identified, as previously described. Here, we also report the activation of Ca²⁺ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca²⁺ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.

Dynamics of the Leaf-Litter Arthropod Fauna Following Fire in a Neotropical Woodland Savanna

2009-11-09T08:00:00Z

Fire is an important agent of disturbance in tropical savannas, but relatively few studies have analyzed how soil-and-litter dwelling arthropods respond to fire disturbance despite the critical role these organisms play in nutrient cycling and other biogeochemical processes. Following the incursion of a fire into a woodland savanna ecological reserve in Central Brazil, we monitored the dynamics of litter-arthropod populations for nearly two years in one burned and one unburned area of the reserve. We also performed a reciprocal transplant experiment to determine the effects of fire and litter type on the dynamics of litter colonization by arthropods. Overall arthropod abundance, the abundance of individual taxa, the richness of taxonomic groups, and the species richness of individual taxa (Formiciade) were lower in the burned site. However, both the ordinal-level composition of the litter arthropod fauna and the species-level composition of the litter ant fauna were not dramatically different in the burned and unburned sites. There is evidence that seasonality of rainfall interacts with fire, as differences in arthropod abundance and diversity were more pronounced in the dry than in the wet season. For many taxa the differences in abundance between burned and unburned sites were maintained even when controlling for litter availability and quality. In contrast, differences in abundance for Collembola, Formicidae, and Thysanoptera were only detected in the unmanipulated samples, which had a lower amount of litter in the burned than in the unburned site throughout most of our study period. Together these results suggest that arthropod density declines in fire-disturbed areas as a result of direct mortality, diminished resources (i.e., reduced litter cover) and less favorable microclimate (i.e., increased litter desiccation due to reduction in tree cover). Although these effects were transitory, there is evidence that the increasingly prevalent fire return interval of only 1–2 years may jeopardize the long-term conservation of litter arthropod communities.

An Association Analysis between Mitochondrial DNA A10398G Polymorphism and Temperament in Japanese Young Adults

2009-11-09T08:00:00Z

The mitochondrial (mt) DNA C5178A and A10398G polymorphisms have been reported to be associated with mental disorders such as bipolar disorder. However, the effects of these polymorphisms on temperament in healthy people are poorly understood. Evaluating healthy subjects can have the advantage of providing new strategies for maintaining psychological health and preventing mental illness. We examined the association between mtDNA polymorphisms and temperament in Japanese students. There was no significant difference in examined temperament when analysed by genotypes, 5178–10398 haplotypes, or sex. The subgroup analysis based on sex indicated that there was an interactive effect of the mtDNA A10398G polymorphism and sex on anxiety and obsession. This finding is preliminary and cannot exclude the possibility of false-positive due to small sample size (144 subjects) and multiple statistical testing. Further studies involving a larger sample size or other ethnic groups are necessary to confirm that mtDNA A10398G polymorphism can be a genetic factor for temperament.

Gemcitabine and Arabinosylcytosin Pharmacogenomics: Genome-Wide Association and Drug Response Biomarkers

2009-11-09T08:00:00Z

Cancer patients show large individual variation in their response to chemotherapeutic agents. Gemcitabine (dFdC) and AraC, two cytidine analogues, have shown significant activity against a variety of tumors. We previously used expression data from a lymphoblastoid cell line-based model system to identify genes that might be important for the two drug cytotoxicity. In the present study, we used that same model system to perform a genome-wide association (GWA) study to test the hypothesis that common genetic variation might influence both gene expression and response to the two drugs. Specifically, genome-wide single nucleotide polymorphisms (SNPs) and mRNA expression data were obtained using the Illumina 550K® HumanHap550 SNP Chip and Affymetrix U133 Plus 2.0 GeneChip, respectively, for 174 ethnically-defined “Human Variation Panel” lymphoblastoid cell lines. Gemcitabine and AraC cytotoxicity assays were performed to obtain IC₅₀ values for the cell lines. We then performed GWA studies with SNPs, gene expression and IC₅₀ of these two drugs. This approach identified SNPs that were associated with gemcitabine or AraC IC₅₀ values and with the expression regulation for 29 genes or 30 genes, respectively. One SNP in IQGAP2 (rs3797418) was significantly associated with variation in both the expression of multiple genes and gemcitabine and AraC IC₅₀. A second SNP in TGM3 (rs6082527) was also significantly associated with multiple gene expression and gemcitabine IC50. To confirm the association results, we performed siRNA knock down of selected genes with expression that was associated with rs3797418 and rs6082527 in tumor cell and the knock down altered gemcitabine or AraC sensitivity, confirming our association study results. These results suggest that the application of GWA approaches using cell-based model systems, when combined with complementary functional validation, can provide insights into mechanisms responsible for variation in cytidine analogue response.

Expression of Telomerase and Telomere Length Are Unaffected by either Age or Limb Regeneration in Danio rerio

2009-11-06T08:00:00Z

Background

The zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood.

Methodology/Principal Findings

We have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay.

Conclusions/Significance

In contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish's ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human.

Rapid and Long-Lasting Increase in Sites for Synapse Assembly during Late-Phase Potentiation in Rat Hippocampal Neurons

2009-11-06T08:00:00Z

Long-term potentiation in hippocampal neurons has stages that correspond to the stages of learning and memory. Early-phase (10–30 min) potentiation is accompanied by rapid increases in clusters or puncta of presynaptic and postsynaptic proteins, which depend on actin polymerization but not on protein synthesis. We have now examined changes in pre- and postsynaptic puncta and structures during glutamate-induced late-phase (3 hr) potentiation in cultured hippocampal neurons. We find that (1) the potentiation is accompanied by long-lasting maintenance of the increases in puncta, which depends on protein synthesis, (2) most of the puncta and synaptic structures are very dynamic, continually assembling and disassembling at sites that are more stable than the puncta or structures themselves, (3) the increase in presynaptic puncta appears to be due to both rapid and more gradual increases in the number of sites where the puncta may form, and also to the stabilization of existing puncta, (4) under control conditions, puncta of postsynaptic proteins behave similarly to puncta of presynaptic proteins and share sites with them, and (5) the increase in presynaptic puncta is accompanied by a similar increase in presumably presynaptic structures, which may form at distinct as well as shared sites. The new sites could contribute to the transition between the early and late phase mechanisms of plasticity by serving as seeds for the formation and maintenance of new synapses, thus acting as local “tags” for protein synthesis-dependent synaptic growth during late-phase plasticity.

Tunable Chemokine Production by Antigen Presenting Dendritic Cells in Response to Changes in Regulatory T Cell Frequency in Mouse Reactive Lymph Nodes

2009-11-06T08:00:00Z

Background

Although evidence exists that regulatory T cells (Tregs) can suppress the effector phase of immune responses, it is clear that their major role is in suppressing T cell priming in secondary lymphoid organs. Recent experiments using two photon laser microscopy indicate that dendritic cells (DCs) are central to Treg cell function and that the in vivo mechanisms of T cell regulation are more complex than those described in vitro.

Principal Findings

Here we have sought to determine whether and how modulation of Treg numbers modifies the lymph node (LN) microenvironment. We found that pro-inflammatory chemokines—CCL2 (MCP-1) and CCL3 (MIP-la)—are secreted in the LN early (24 h) after T cell activation, that this secretion is dependent on antigen-specific DC–T cell interactions, and that it was inversely related to the frequency of Tregs specific for the same antigen. Furthermore, we demonstrate that Tregs modify the chemoattractant properties of antigen-presenting DCs, which, as the frequency of Tregs increases, fail to produce CCL2 and CCL3 and to attract antigen-specific T cells.

Conclusions

These results substantiate a major role of Tregs in LN patterning during antigen-specific immune responses.

Inaccurate Diagnosis of HIV-1 Group M and O Is a Key Challenge for Ongoing Universal Access to Antiretroviral Treatment and HIV Prevention in Cameroon

2009-11-06T08:00:00Z

Background

Increased access to HIV testing is essential in working towards universal access to HIV prevention and treatment in resource-limited countries. We here evaluated currently used HIV diagnostic tests and algorithms in Cameroon for their ability to correctly identify HIV infections.

Methods

We estimated sensitivity, specificity, and positive and negative predictive values of 5 rapid/simple tests, of which 3 were used by the national program, and 2 fourth generation ELISAs. The reference panel included 500 locally collected samples; 187 HIV -1 M, 10 HIV-1 O, 259 HIV negative and 44 HIV indeterminate plasmas.

Results

None of the 5 rapid assays and only 1 ELISA reached the current WHO/UNAIDS recommendations on performance of HIV tests of at least 99% sensitivity and 98% specificity. Overall, sensitivities ranged between 94.1% and 100%, while specificities were 88.0% to 98.8%. The combination of all assays generated up to 9% of samples with indeterminate HIV status, because they reacted discordantly with at least one of the different tests. Including HIV indeterminate samples in test efficiency calculations significantly decreased specificities to a range from 77.9% to 98.0%. Finally, two rapid assays failed to detect all HIV-1 group O variants tested, with one rapid test detecting only 2 out of 10 group O specimens.

Conclusion

In the era of ART scaling-up in Africa, significant proportions of false positive but also false negative results are still observed with HIV screening tests commonly used in Africa, resulting in inadequate treatment and prevention strategies. Depending on tests or algorithms used, up to 6% of HIV-1 M and 80% of HIV-1 O infected patients in Cameroon do not receive ART and adequate counseling to prevent further transmission due to low sensitivities. Also, the use of tests with low specificities could imply inclusion of up to 12% HIV negative people in ART programs and increase budgets in addition to inconveniences caused to patients.

Roles of ES Cell-Derived Gliogenic Neural Stem/Progenitor Cells in Functional Recovery after Spinal Cord Injury

2009-11-06T08:00:00Z

Transplantation of neural stem/progenitor cells (NS/PCs) following the sub-acute phase of spinal cord injury (SCI) has been shown to promote functional recovery in rodent models. However, the types of cells most effective for treating SCI have not been clarified. Taking advantage of our recently established neurosphere-based culture system of ES cell-derived NS/PCs, in which primary neurospheres (PNS) and passaged secondary neurospheres (SNS) exhibit neurogenic and gliogenic potentials, respectively, here we examined the distinct effects of transplanting neurogenic and gliogenic NS/PCs on the functional recovery of a mouse model of SCI. ES cell-derived PNS and SNS transplanted 9 days after contusive injury at the Th10 level exhibited neurogenic and gliogenic differentiation tendencies, respectively, similar to those seen in vitro. Interestingly, transplantation of the gliogenic SNS, but not the neurogenic PNS, promoted axonal growth, remyelination, and angiogenesis, and resulted in significant locomotor functional recovery after SCI. These findings suggest that gliogenic NS/PCs are effective for promoting the recovery from SCI, and provide essential insight into the mechanisms through which cellular transplantation leads to functional improvement after SCI.

Current Status of a Model System: The Gene Gp-9 and Its Association with Social Organization in Fire Ants

2009-11-06T08:00:00Z

The Gp-9 gene in fire ants represents an important model system for studying the evolution of social organization in insects as well as a rich source of information relevant to other major evolutionary topics. An important feature of this system is that polymorphism in social organization is completely associated with allelic variation at Gp-9, such that single-queen colonies (monogyne form) include only inhabitants bearing B-like alleles while multiple-queen colonies (polygyne form) additionally include inhabitants bearing b-like alleles. A recent study of this system by Leal and Ishida (2008) made two major claims, the validity and significance of which we examine here. After reviewing existing literature, analyzing the methods and results of Leal and Ishida (2008), and generating new data from one of their study sites, we conclude that their claim that polygyny can occur in Solenopsis invicta in the U.S.A. in the absence of expression of the b-like allele Gp-9^b is unfounded. Moreover, we argue that available information on insect OBPs (the family of proteins to which GP-9 belongs), on the evolutionary/population genetics of Gp-9, and on pheromonal/behavioral control of fire ant colony queen number fails to support their view that GP-9 plays no role in the chemosensory-mediated communication that underpins regulation of social organization. Our analyses lead us to conclude that there are no new reasons to question the existing consensus view of the Gp-9 system outlined in Gotzek and Ross (2007).

Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy

2009-11-06T08:00:00Z

The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.

Genetic Diversity of Arginine Catabolic Mobile Element in Staphylococcus epidermidis

2009-11-06T08:00:00Z

Background

The methicillin-resistant Staphylococcus aureus clone USA300 contains a novel mobile genetic element, arginine catabolic mobile element (ACME), that contributes to its enhanced capacity to grow and survive within the host. Although ACME appears to have been transferred into USA300 from S. epidermidis, the genetic diversity of ACME in the latter species remains poorly characterized.

Methodology/Principal Findings

To assess the prevalence and genetic diversity of ACME, 127 geographically diverse S. epidermidis isolates representing 86 different multilocus sequence types (STs) were characterized. ACME was found in 51% (65/127) of S. epidermidis isolates. The vast majority (57/65) of ACME-containing isolates belonged to the predominant S. epidermidis clonal complex CC2. ACME was often found in association with different allotypes of staphylococcal chromosome cassette mec (SCCmec) which also encodes the recombinase function that facilities mobilization ACME from the S. epidermidis chromosome. Restriction fragment length polymorphism, PCR scanning and DNA sequencing allowed for identification of 39 distinct ACME genetic variants that differ from one another in gene content, thereby revealing a hitherto uncharacterized genetic diversity within ACME. All but one ACME variants were represented by a single S. epidermidis isolate; the singular variant, termed ACME-I.02, was found in 27 isolates, all of which belonged to the CC2 lineage. An evolutionary model constructed based on the eBURST algorithm revealed that ACME-I.02 was acquired at least on 15 different occasions by strains belonging to the CC2 lineage.

Conclusions/Significance

ACME-I.02 in diverse S. epidermidis isolates were nearly identical in sequence to the prototypical ACME found in USA300 MRSA clone, providing further evidence for the interspecies transfer of ACME from S. epidermidis into USA300.

Penicillin Induced Persistence in Chlamydia trachomatis: High Quality Time Lapse Video Analysis of the Developmental Cycle

2009-11-06T08:00:00Z

Background

Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.

Principal Findings

Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.

Conclusions

We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs.

A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients

2009-11-06T08:00:00Z

The aim of this study was to search for lipid signatures in blood plasma from cystic fibrosis (CF) patients using a novel MALDI-TOF-ClinProTools™ strategy, initially developed for protein analysis, and thin layer chromatography coupled to MALDI-TOF (TLC-MALDI). Samples from 33 CF patients and 18 healthy children were subjected to organic extraction and column chromatography separation of lipid classes. Extracts were analyzed by MALDI-TOF, ion signatures were compared by the ClinProTools™ software and by parallel statistical analyses. Relevant peaks were identified by LC-MSn. The ensemble of analyses provided 11 and 4 peaks differentially displayed in CF vs healthy and in mild vs severe patients respectively. Ten ions were significantly decreased in all patients, corresponding to 4 lysophosphatidylcholine (18∶0, 18∶2, 20∶3, and 20∶5) and 6 phosphatidylcholine (36∶5, O-38∶0, 38∶4, 38∶5, 38∶6, and P-40∶1) species. One sphingolipid, SM(d18∶0), was significantly increased in all patients. Four PC forms (36∶3, 36∶5, 38∶5, and 38∶6) were consistently downregulated in severe vs mild patients. These observations were confirmed by TLC-MALDI. These results suggest that plasma phospholipid signatures may be able to discriminate mild and severe forms of CF, and show for the first time MALDI-TOF-ClinProTools™ as a suitable methodology for the search of lipid markers in CF.

A Comprehensive Sequence and Disease Correlation Analyses for the C-Terminal Region of CagA Protein of Helicobacter pylori

2009-11-06T08:00:00Z

Chronic Helicobacter pylori infection is known to be associated with the development of peptic ulcer, gastric cancer and gastric lymphoma. Currently, the bacterial factors of H. pylori are reported to be important in the development of gastroduodenal diseases. CagA protein, encoded by the cagA, is the best studied virulence factor of H. pylori. The pathogenic CagA protein contains a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal. This repeat region is reported to be involved in the pathogenesis of gastroduodenal diseases. The segments containing EPIYA motifs have been designated as segments A, B, C, and D; however the classification and disease relation are still unclear. This study used 560 unique CagA sequences containing 1,796 EPIYA motifs collected from public resources, including 274 Western and 286 East Asian strains with clinical data obtained from 433 entries. Fifteen types of EPIYA or EPIYA-like sequences are defined. In addition to four previously reported major segment types, several minor segment types (e.g., segment B′, B′′) and more than 30 sequence types (e.g., ABC, ABD) were defined using our classification method. We confirm that the sequences from Western and East Asian strains contain segment C and D, respectively. We also confirm that strains with two EPIYA segment C have a greater chance of developing gastric cancer than those with one segment C. Our results shed light on the relationships between the types of CagAs, the country of origin of each sequence type, and the frequency of gastric disease.

Islet Formation during the Neonatal Development in Mice

2009-11-06T08:00:00Z

The islet of Langerhans is a unique micro-organ within the exocrine pancreas, which is composed of insulin-secreting beta-cells, glucagon-secreting alpha-cells, somatostatin-secreting delta-cells, pancreatic polypeptide-secreting PP cells and ghrelin-secreting epsilon-cells. Islets also contain non-endocrine cell types such as endothelial cells. However, the mechanism(s) of islet formation is poorly understood due to technical difficulties in capturing this dynamic event in situ. We have developed a method to monitor beta-cell proliferation and islet formation in the intact pancreas using transgenic mice in which the beta-cells are specifically tagged with a fluorescent protein. Endocrine cells proliferate contiguously, forming branched cord-like structures in both embryos and neonates. Our study has revealed long stretches of interconnected islets located along large blood vessels in the neonatal pancreas. Alpha-cells span the elongated islet-like structures, which we hypothesize represent sites of fission and facilitate the eventual formation of discrete islets. We propose that islet formation occurs by a process of fission following contiguous endocrine cell proliferation, rather than by local aggregation or fusion of isolated beta-cells and islets. Mathematical modeling of the fission process in the neonatal islet formation is also presented.

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

2009-11-06T08:00:00Z

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.

Inhibition of Neuraminidase Inhibitor-Resistant Influenza Virus by DAS181, a Novel Sialidase Fusion Protein

2009-11-06T08:00:00Z

Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase®), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

2009-11-05T08:00:00Z

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.

IgG Antibodies against Measles, Rubella, and Varicella Zoster Virus Predict Conversion to Multiple Sclerosis in Clinically Isolated Syndrome

2009-11-05T08:00:00Z

Background

Multiple sclerosis (MS) is characterized by a polyspecific B-cell response to neurotropic viruses such as measles, rubella and varicella zoster, with the corresponding antibodies measurable in CSF as the so-called “MRZ reaction” (MRZR). We aimed to evaluate the relevance of MRZR to predict conversion of patients with clinically isolated syndrome (CIS) to MS, and to compare it to oligoclonal bands (OCB) and MRI.

Methodology/Principal Findings

MRZR was determined in a prospective study over 2 years including 40 patients that remained CIS over follow-up (CIS-CIS) and 49 patients that developed MS (CIS-RRMS) using ELISA. Using logistic regression, a score (MRZS) balancing the predictive value of the antibody indices included in MRZR was defined (9 points measles, 8 points rubella, 1 point varicella zoster, cutpoint: sum of scores greater 10).

MRZR and MRZS were significantly more frequent in CIS-RRMS as compared to CIS-CIS (p = 0.04 and p = 0.02). MRZS showed the best positive predictive value (PPV) of all parameters investigated (79%, 95%-CI: 54–94%), which could be further increased by combination with MRI (91%, 95%-CI: 59–99%).

Conclusions/Significance

Our data indicate the relevance of MRZR to predict conversion to MS. It furthermore shows the importance of weighting the different antibody indices included in MRZR and suggest that patients with positive MRZR are candidates for an early begin of immunomodulatory therapy.

Correlating Molecular Phylogeny with Venom Apparatus Occurrence in Panamic Auger Snails (Terebridae)

2009-11-05T08:00:00Z

Central to the discovery of neuroactive compounds produced by predatory marine snails of the superfamily Conoidea (cone snails, terebrids, and turrids) is identifying those species with a venom apparatus. Previous analyses of western Pacific terebrid specimens has shown that some Terebridae groups have secondarily lost their venom apparatus. In order to efficiently characterize terebrid toxins, it is essential to devise a key for identifying which species have a venom apparatus. The findings presented here integrate molecular phylogeny and the evolution of character traits to infer the presence or absence of the venom apparatus in the Terebridae. Using a combined dataset of 156 western and 33 eastern Pacific terebrid samples, a phylogenetic tree was constructed based on analyses of 16S, COI and 12S mitochondrial genes. The 33 eastern Pacific specimens analyzed represent four different species: Acus strigatus, Terebra argyosia, T. ornata, and T. cf. formosa. Anatomical analysis was congruent with molecular characters, confirming that species included in the clade Acus do not have a venom apparatus, while those in the clade Terebra do. Discovery of the association between terebrid molecular phylogeny and the occurrence of a venom apparatus provides a useful tool for effectively identifying the terebrid lineages that may be investigated for novel pharmacological active neurotoxins, enhancing conservation of this important resource, while providing supplementary information towards understanding terebrid evolutionary diversification.

Visual Properties of Transgenic Rats Harboring the Channelrhodopsin-2 Gene Regulated by the Thy-1.2 Promoter

2009-11-05T08:00:00Z

Channelrhodopsin-2 (ChR2), one of the archea-type rhodopsins from green algae, is a potentially useful optogenetic tool for restoring vision in patients with photoreceptor degeneration, such as retinitis pigmentosa. If the ChR2 gene is transferred to retinal ganglion cells (RGCs), which send visual information to the brain, the RGCs may be repurposed to act as photoreceptors. In this study, by using a transgenic rat expressing ChR2 specifically in the RGCs under the regulation of a Thy-1.2 promoter, we tested the possibility that direct photoactivation of RGCs could restore effective vision. Although the contrast sensitivities of the optomotor responses of transgenic rats were similar to those observed in the wild-type rats, they were enhanced for visual stimuli of low-spatial frequency after the degeneration of native photoreceptors. This result suggests that the visual signals derived from the ChR2-expressing RGCs were reinterpreted by the brain to form behavior-related vision.

Effect before Cause: Supramodal Recalibration of Sensorimotor Timing

2009-11-05T08:00:00Z

Background

Our motor actions normally generate sensory events, but how do we know which events were self generated and which have external causes? Here we use temporal adaptation to investigate the processing stage and generality of our sensorimotor timing estimates.

Methodology/Principal Findings

Adaptation to artificially-induced delays between action and event can produce a startling percept—upon removal of the delay it feels as if the sensory event precedes its causative action. This temporal recalibration of action and event occurs in a quantitatively similar manner across the sensory modalities. Critically, it is robust to the replacement of one sense during the adaptation phase with another sense during the test judgment.

Conclusions/Significance

Our findings suggest a high-level, supramodal recalibration mechanism. The effects are well described by a simple model which attempts to preserve the expected synchrony between action and event, but only when causality indicates it is reasonable to do so. We further demonstrate that this model successfully characterises related adaptation data from outside the sensorimotor domain.

Deep Sequencing of Viroid-Derived Small RNAs from Grapevine Provides New Insights on the Role of RNA Silencing in Plant-Viroid Interaction

2009-11-05T08:00:00Z

Background

Viroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21–24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms.

Methodology/Principal Findings

To gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (−) viroid RNAs, with a prevalence of those from the (−) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots.

Conclusions/Significance

These findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression.

Mechanism of Dinitrochlorobenzene-Induced Dermatitis in Mice: Role of Specific Antibodies in Pathogenesis

2009-11-05T08:00:00Z

Background

Dinitrochlorobenzene-induced contact hypersensitivity is widely considered as a cell-mediated rather than antibody-mediated immune response. At present, very little is known about the role of antigen-specific antibodies and B cells in the development of dinitrochlorobenzene-induced hypersensitivity reactions, and this is the subject of the present investigation.

Methodology/Principal Findings

Data obtained from multiple lines of experiments unequivocally showed that the formation of dinitrochlorobenzene-specific Abs played an important role in the development of dinitrochlorobenzene-induced contact hypersensitivity. The appearance of dinitrochlorobenzene-induced skin dermatitis matched in timing the appearance of the circulating dinitrochlorobenzene-specific antibodies. Adoptive transfer of sera containing dinitrochlorobenzene-specific antibodies from dinitrochlorobenzene-treated mice elicited a much stronger hypersensitivity reaction than the adoptive transfer of lymphocytes from the same donors. Moreover, dinitrochlorobenzene-induced contact hypersensitivity was strongly suppressed in B cell-deficient mice with no DNCB-specific antibodies. It was also observed that treatment of animals with dinitrochlorobenzene polarized Th cells into Th2 differentiation by increasing the production of Th2 cytokines while decreasing the production of Th1 cytokines.

Conclusions/Significance

In striking contrast to the long-held belief that dinitrochlorobenzene-induced contact hypersensitivity is a cell-mediated immune response, the results of our present study demonstrated that the production of dinitrochlorobenzene-specific antibodies by activated B cells played an indispensible role in the pathogenesis of dinitrochlorobenzene-induced CHS. These findings may provide new possibilities in the treatment of human contact hypersensitivity conditions.

A Continuum of Cell States Spans Pluripotency and Lineage Commitment in Human Embryonic Stem Cells

2009-11-05T08:00:00Z

Background

Commitment in embryonic stem cells is often depicted as a binary choice between alternate cell states, pluripotency and specification to a particular germ layer or extraembryonic lineage. However, close examination of human ES cell cultures has revealed significant heterogeneity in the stem cell compartment.

Methodology/Principal Findings

We isolated subpopulations of embryonic stem cells using surface markers, then examined their expression of pluripotency genes and lineage specific transcription factors at the single cell level, and tested their ability to regenerate colonies of stem cells. Transcript analysis of single embryonic stem cells showed that there is a gradient and a hierarchy of expression of pluripotency genes in the population. Even cells at the top of the hierarchy generally express only a subset of the stem cell genes studied. Many cells co-express pluripotency and lineage specific genes. Cells along the continuum show a progressively decreasing likelihood of self renewal as their expression of stem cell surface markers and pluripotency genes wanes. Most cells that are positive for stem cell surface markers express Oct-4, but only those towards the top of the hierarchy express the nodal receptor TDGF-1 and the growth factor GDF3.

Significance

These findings on gene expression in single embryonic stem cells are in concert with recent studies of early mammalian development, which reveal molecular heterogeneity and a stochasticity of gene expression in blastomeres. Our work indicates that only a small fraction of the population resides at the top of the hierarchy, that lineage priming (co-expression of stem cell and lineage specific genes) characterizes pluripotent stem cell populations, and that extrinsic signaling pathways are upstream of transcription factor networks that control pluripotency.

P-Loop Residues Critical for Selectivity in K⁺ Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

2009-11-05T08:00:00Z

HCN channels are thought to be structurally similar to K_v channels, but show much lower selectivity for K⁺. The ~3.3 Å selectivity filter of K⁺ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L₄₇₈C₄₇₉IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K⁺ channels, we also studied C479T channels. Based on permeability ratios (P_X/P_K), C479T HCN4 channels (K⁺(1)>Rb⁺(0.85)>Cs⁺(0.59)>Li⁺(0.50)≥Na⁺(0.49)) were less selective than WT rabbit HCN4 (K⁺(1)>Rb⁺(0.48)>Cs⁺(0.31)≥Na⁺(0.29)>Li⁺(0.03)), indicating that the TIGYG sequence is insufficient to confer K⁺ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K⁺ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K⁺ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K⁺ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K⁺ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K⁺ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K⁺ channels originate from differences outside the P-loop region.

A Role for Immune Responses against Non-CS Components in the Cross-Species Protection Induced by Immunization with Irradiated Malaria Sporozoites

2009-11-05T08:00:00Z

Immunization with irradiated Plasmodium sporozoites induces sterile immunity in rodents, monkeys and humans. The major surface component of the sporozoite the circumsporozoite protein (CS) long considered as the antigen predominantly responsible for this immunity, thus remains the leading candidate antigen for vaccines targeting the parasite's pre-erythrocytic (PE) stages. However, this role for CS was questioned when we recently showed that immunization with irradiated sporozoites (IrrSpz) of a P. berghei line whose endogenous CS was replaced by that of P. falciparum still conferred sterile protection against challenge with wild type P. berghei sporozoites. In order to investigate the involvement of CS in the cross-species protection recently observed between the two rodent parasites P. berghei and P. yoelii, we adopted our gene replacement approach for the P. yoelii CS and exploited the ability to conduct reciprocal challenges. Overall, we found that immunization led to sterile immunity irrespective of the origin of the CS in the immunizing or challenge sporozoites. However, for some combinations, immune responses to CS contributed to the acquisition of protective immunity and were dependent on the immunizing IrrSpz dose. Nonetheless, when data from all the cross-species immunization/challenges were considered, the immune responses directed against non-CS parasite antigens shared by the two parasite species played a major role in the sterile protection induced by immunization with IrrSpz. This opens the perspective to develop a single vaccine formulation that could protect against multiple parasite species.

Human Blood Vessel–Derived Endothelial Progenitors for Endothelialization of Small Diameter Vascular Prosthesis

2009-11-05T08:00:00Z

Background

Coronary bypass graft failure as a result of acute thrombosis and intimal hyperplasia has been the major challenge in surgical procedures involving small-diameter vascular prosthesis. Coating synthetic grafts with patients' own endothelial cells has been suggested to improve the patency rate and overall success of bypass surgeries.

Methodology/Principal Findings

We isolated endothelial progenitor cells (EPCs) from leftover pieces of human saphenous vein/mammary artery. We demonstrate that EPCs can be expanded to generate millions of cells under low-density culture conditions. Exposure to high-density conditions induces differentiation to endothelial cell phenotype. EPC–derived endothelial cells show expression of CD144^high, CD31, and vWF. We then assessed the ability of differentiated endothelial cells to adhere and grow on small diameter expanded polytetrafluoroethylene (ePTFE) tubings. Since ePTFE tubings are highly hydrophobic, we optimized protocols to introduce hydrophilic groups on luminal surface of ePTFE tubings. We demonstrate here a stepwise protocol that involves introduction of hydrophilic moieties and coating with defined ECM components that support adhesion of endothelial cells, but not of blood platelets.

Conclusion/Significance

Our data confirms that endothelial progenitors obtained from adult human blood vessels can be expanded in vitro under xenoprotein-free conditions, for potential use in endothelialization of small diameter ePTFE grafts. These endothelialized grafts may represent a promising treatment strategy for improving the clinical outcome of small-caliber vascular grafts in cardiac bypass surgeries.

A Decade Later, How Much of Rwanda's Musculoskeletal Impairment Is Caused by the War in 1994 and by Related Violence?

2009-11-05T08:00:00Z

Background

In 1994 there was a horrific genocide in Rwanda following years of tension, resulting in the murder of at least 800,000 people. Although many people were injured in addition to those killed, no attempt has been made to assess the lasting burden of physical injuries related to these events. The aim of this study was to estimate the current burden of musculoskeletal impairment (MSI) attributable to the 1994 war and related violence.

Methodology/Principal Findings

A national cross-sectional survey of MSI was conducted in Rwanda. 105 clusters of 80 people were selected through probability proportionate to size sampling. Households within clusters were selected through compact segment sampling. Enumerated people answered a seven-question screening test to assess whether they might have an MSI. Those who were classed as potential cases in the screening test were examined and interviewed by a physiotherapist, using a standard protocol that recorded the site, nature, cause, and severity of the MSI. People with MSI due to trauma were asked whether this trauma occurred during the 1990–1994 war or during the episodes that preceded or followed this war. Out of 8,368 people enumerated, 6,757 were available for screening and examination (80.8%). 352 people were diagnosed with an MSI (prevalence = 5.2%, 95% CI = 4.5–5.9%). 106 cases of MSI (30.6%) were classified as resulting from trauma, based on self-report and the physiotherapist's assessment. Of these, 14 people (13.2%) reported that their trauma-related MSI occurred during the 1990–1994 war, and a further 7 (6.6%) that their trauma-related MSI occurred during the violent episodes that preceded and followed the war, giving an overall prevalence of trauma-related MSI related to the 1990–1994 war of 0.3% (95% CI = 0.2–0.4%).

Conclusions/Significance

A decade on, the overall prevalence of MSI was relatively high in Rwanda but few cases appeared to be the result of the 1994 war or related violence.

Improvements of Sound Localization Abilities by the Facial Ruff of the Barn Owl (Tyto alba) as Demonstrated by Virtual Ruff Removal

2009-11-05T08:00:00Z

Background

When sound arrives at the eardrum it has already been filtered by the body, head, and outer ear. This process is mathematically described by the head-related transfer functions (HRTFs), which are characteristic for the spatial position of a sound source and for the individual ear. HRTFs in the barn owl (Tyto alba) are also shaped by the facial ruff, a specialization that alters interaural time differences (ITD), interaural intensity differences (ILD), and the frequency spectrum of the incoming sound to improve sound localization. Here we created novel stimuli to simulate the removal of the barn owl's ruff in a virtual acoustic environment, thus creating a situation similar to passive listening in other animals, and used these stimuli in behavioral tests.

Methodology/Principal Findings

HRTFs were recorded from an owl before and after removal of the ruff feathers. Normal and ruff-removed conditions were created by filtering broadband noise with the HRTFs. Under normal virtual conditions, no differences in azimuthal head-turning behavior between individualized and non-individualized HRTFs were observed. The owls were able to respond differently to stimuli from the back than to stimuli from the front having the same ITD. By contrast, such a discrimination was not possible after the virtual removal of the ruff. Elevational head-turn angles were (slightly) smaller with non-individualized than with individualized HRTFs. The removal of the ruff resulted in a large decrease in elevational head-turning amplitudes.

Conclusions/Significance

The facial ruff a) improves azimuthal sound localization by increasing the ITD range and b) improves elevational sound localization in the frontal field by introducing a shift of iso–ILD lines out of the midsagittal plane, which causes ILDs to increase with increasing stimulus elevation. The changes at the behavioral level could be related to the changes in the binaural physical parameters that occurred after the virtual removal of the ruff. These data provide new insights into the function of external hearing structures and open up the possibility to apply the results on autonomous agents, creation of virtual auditory environments for humans, or in hearing aids.

Timing Constraints of In Vivo Gag Mutations during Primary HIV-1 Subtype C Infection

2009-11-05T08:00:00Z

Background

Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection.

Methods

A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations.

Results

Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes.

Conclusions

The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness.

Common Polymorphisms Influencing Serum Uric Acid Levels Contribute to Susceptibility to Gout, but Not to Coronary Artery Disease

2009-11-05T08:00:00Z

Background

Recently, a large meta-analysis including over 28,000 participants identified nine different loci with association to serum uric acid (UA) levels. Since elevated serum UA levels potentially cause gout and are a possible risk factor for coronary artery disease (CAD) and myocardial infarction (MI), we performed two large case-control association analyses with participants from the German MI Family Study. In the first study, we assessed the association of the qualitative trait gout and ten single nucleotide polymorphisms (SNP) markers that showed association to UA serum levels. In the second study, the same genetic polymorphisms were analyzed for association with CAD.

Methods and Findings

A total of 683 patients suffering from gout and 1,563 healthy controls from the German MI Family Study were genotyped. Nine SNPs were identified from a recently performed genome-wide meta-analysis on serum UA levels (rs12129861, rs780094, rs734553, rs2231142, rs742132, rs1183201, rs12356193, rs17300741 and rs505802). Additionally, the marker rs6855911 was included which has been associated with gout in our cohort in a previous study. SNPs rs734553 and rs6855911, located in SLC2A9, and SNP rs2231142, known to be a missense polymorphism in ABCG2, were associated with gout (p = 5.6*10⁻⁷, p = 1.1*10⁻⁷, and p = 1.3*10⁻³, respectively). Other SNPs in the genes PDZK1, GCKR, LRRC16A, SLC17A1-SLC17A3, SLC16A9, SLC22A11 and SLC22A12 failed the significance level. None of the ten markers were associated with risk to CAD in our study sample of 1,473 CAD cases and 1,241 CAD-free controls.

Conclusion

SNP markers in SLC2A9 and ABCG2 genes were found to be strongly associated with the phenotype gout. However, not all SNP markers influencing serum UA levels were also directly associated with the clinical manifestation of gout in our study sample. In addition, none of these SNPs showed association with the risk to CAD in the German MI Family Study.

MALDI Profiling of Human Lung Cancer Subtypes

2009-11-05T08:00:00Z

Background

Proteomics is expected to play a key role in cancer biomarker discovery. Although it has become feasible to rapidly analyze proteins from crude cell extracts using mass spectrometry, complex sample composition hampers this type of measurement. Therefore, for effective proteome analysis, it becomes critical to enrich samples for the analytes of interest. Despite that one-third of the proteins in eukaryotic cells are thought to be phosphorylated at some point in their life cycle, only a low percentage of intracellular proteins is phosphorylated at a given time.

Methodology/Principal Findings

In this work, we have applied chromatographic phosphopeptide enrichment techniques to reduce the complexity of human clinical samples. A novel method for high-throughput peptide profiling of human tumor samples, using Parallel IMAC and MALDI-TOF MS, is described. We have applied this methodology to analyze human normal and cancer lung samples in the search for new biomarkers. Using a highly reproducible spectral processing algorithm to produce peptide mass profiles with minimal variability across the samples, lineal discriminant-based and decision tree–based classification models were generated. These models can distinguish normal from tumor samples, as well as differentiate the various non–small cell lung cancer histological subtypes.

Conclusions/Significance

A novel, optimized sample preparation method and a careful data acquisition strategy is described for high-throughput peptide profiling of small amounts of human normal lung and lung cancer samples. We show that the appropriate combination of peptide expression values is able to discriminate normal lung from non-small cell lung cancer samples and among different histological subtypes. Our study does emphasize the great potential of proteomics in the molecular characterization of cancer.

Reduced Physiological Complexity in Robust Elderly Adults with the APOE ε4 Allele

2009-11-05T08:00:00Z

Background

It is unclear whether the loss of physiological complexity during the aging process is due to genetic variations. The APOE gene has been studied extensively in regard to its relationship with aging-associated medical illness. We hypothesize that diminished physiological complexity, as measured by heart rate variability, is influenced by polymorphisms in the APOE allele among elderly individuals.

Methodology/Principal Findings

A total of 102 robust, non-demented, elderly subjects with normal functions of daily activities participated in this study (97 males and 5 females, aged 79.2±4.4 years, range 72–92 years). Among these individuals, the following two APOE genotypes were represented: ε4 non-carriers (n = 87, 85.3%) and ε4 carriers (n = 15, 14.7%). Multi-scale entropy (MSE), an analysis used in quantifying complexity for nonlinear time series, was employed to analyze heart-rate dynamics. Reduced physiological complexity, as measured by MSE, was significantly associated with the presence of the APOE ε4 allele in healthy elderly subjects, as compared to APOE ε4 allele non-carriers (24.6±5.5 versus 28.9±5.2, F = 9.429, p = 0.003, respectively).

Conclusions/Significance

This finding suggests a role for the APOE gene in the diminished physiological complexity seen in elderly populations.

Conditional Tek Promoter-Driven Deletion of Arginyltransferase in the Germ Line Causes Defects in Gametogenesis and Early Embryonic Lethality in Mice

2009-11-05T08:00:00Z

Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis—capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene—a problem that has not previously been reported for this commercial mouse strain—a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.

A Modeling Framework to Describe the Transmission of Bluetongue Virus within and between Farms in Great Britain

2009-11-05T08:00:00Z

Background

Recently much attention has been given to developing national-scale micro-simulation models for livestock diseases that can be used to predict spread and assess the impact of control measures. The focus of these models has been on directly transmitted infections with little attention given to vector-borne diseases such as bluetongue, a viral disease of ruminants transmitted by Culicoides biting midges. Yet BT has emerged over the past decade as one of the most important diseases of livestock.

Methodology/Principal Findings

We developed a stochastic, spatially-explicit, farm-level model to describe the spread of bluetongue virus (BTV) within and between farms. Transmission between farms was modeled by a generic kernel, which includes both animal and vector movements. Once a farm acquired infection, the within-farm dynamics were simulated based on the number of cattle and sheep kept on the farm and on local temperatures. Parameter estimates were derived from the published literature and using data from the outbreak of bluetongue in northern Europe in 2006. The model was validated using data on the spread of BTV in Great Britain during 2007. The sensitivity of model predictions to the shape of the transmission kernel was assessed.

Conclusions/Significance

The model is able to replicate the dynamics of BTV in Great Britain. Although uncertainty remains over the precise shape of the transmission kernel and certain aspects of the vector, the modeling approach we develop constitutes an ideal framework in which to incorporate these aspects as more and better data become available. Moreover, the model provides a tool with which to examine scenarios for the spread and control of BTV in Great Britain.

Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy

2009-11-05T08:00:00Z

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.

The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

2009-11-05T08:00:00Z

Background

RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic β-cells and analyzed the impact on different steps of the insulin-secretory process.

Methodology/Principal Findings

We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3–5 min and reaches a plateau after 10–15 min. The activation of the GTPase is triggered by increases in intracellular Ca²⁺ and cAMP and is prevented by the L-type voltage-gated Ca²⁺ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane.

Conclusions/Significance

Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca²⁺ and cAMP controls hormone release from pancreatic β-cell by coordinating the execution of different events in the secretory pathway.

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

2009-11-05T08:00:00Z

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion.

Inducible Costimulator Expression Regulates the Magnitude of Th2-Mediated Airway Inflammation by Regulating the Number of Th2 Cells

2009-11-04T08:00:00Z

Background

Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5′ promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology.

Methodology/Principal Findings

We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS^+/+ and ICOS^+/− mice in a Th2 model of airway inflammation, we found that T cells from the ICOS^+/− mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS^+/− mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS^+/− is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4.

Conclusion

These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.

Gemini, a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression

2009-11-04T08:00:00Z

Background

The development of collections of quantitatively characterized standard biological parts should facilitate the engineering of increasingly complex and novel biological systems. The existing enzymatic and fluorescent reporters that are used to characterize biological part functions exhibit strengths and limitations. Combining both enzymatic and fluorescence activities within a single reporter protein would provide a useful tool for biological part characterization.

Methodology/Principal Findings

Here, we describe the construction and quantitative characterization of Gemini, a fusion between the β-galactosidase (β-gal) α-fragment and the N-terminus of full-length green fluorescent protein (GFP). We show that Gemini exhibits functional β-gal activity, which we assay with plates and fluorometry, and functional GFP activity, which we assay with fluorometry and microscopy. We show that the protein fusion increases the sensitivity of β-gal activity and decreases the sensitivity of GFP.

Conclusions/Significance

Gemini is therefore a bifunctional reporter with a wider dynamic range than the β-gal α-fragment or GFP alone. Gemini enables the characterization of gene expression, screening assays via enzymatic activity, and quantitative single-cell microscopy or FACS via fluorescence activity. The analytical flexibility afforded by Gemini will likely increase the efficiency of research, particularly for screening and characterization of libraries of standard biological parts.

The Transcriptome of the Nosocomial Pathogen Enterococcus faecalis V583 Reveals Adaptive Responses to Growth in Blood

2009-11-04T08:00:00Z

Background

Enterococcus faecalis plays a dual role in human ecology, predominantly existing as a commensal in the alimentary canal, but also as an opportunistic pathogen that frequently causes nosocomial infections like bacteremia. A number of virulence factors that contribute to the pathogenic potential of E. faecalis have been established. However, the process in which E. faecalis gains access to the bloodstream and establishes a persistent infection is not well understood.

Methodology/Principal Findings

To enhance our understanding of how this commensal bacterium adapts during a bloodstream infection and to examine the interplay between genes we designed an in vitro experiment using genome-wide microarrays to investigate what effects the presence of and growth in blood have on the transcriptome of E. faecalis strain V583. We showed that growth in both 2xYT supplemented with 10% blood and in 100% blood had a great impact on the transcription of many genes in the V583 genome. We identified several immediate changes signifying cellular processes that might contribute to adaptation and growth in blood. These include modulation of membrane fatty acid composition, oxidative and lytic stress protection, acquisition of new available substrates, transport functions including heme/iron transporters and genes associated with virulence in E. faecalis.

Conclusions/Significance

The results presented here reveal that cultivation of E. faecalis in blood in vitro has a profound impact on its transcriptome, which includes a number of virulence traits. Observed regulation of genes and pathways revealed new insight into physiological features and metabolic capacities which enable E. faecalis to adapt and grow in blood. A number of the regulated genes might potentially be useful candidates for development of new therapeutic approaches for treatment of E. faecalis infections.

Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP⁺¹ Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer

2009-11-04T08:00:00Z

The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP⁺¹ antibody. We previously reported that GFAP⁺¹ was expressed in astrocytes and in degenerating neurons in Alzheimer's disease brains. In this study we aimed at further investigating the neuronal GFAP⁺¹ expression and we started by affinity purifying the GFAP⁺¹ antibody. The purified antibody resulted in a loss of neuronal GFAP⁺¹ signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP⁺¹ expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP⁺¹ antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP⁺¹ antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.

Experience Matters: Females Use Smell to Select Experienced Males for Paternal Care

2009-11-04T08:00:00Z

Mate choice and mating preferences often rely on the information content of signals exchanged between potential partners. In species where a female's reproduction is the terminal event in life it is to be expected that females choose high quality males and assess males using some honest indicator of male quality. The Nereidid polychaete, Neanthes acuminata, exhibits monogamous pairing and the release of eggs by females terminates her life and larval success relies entirely on a male's ability to provide paternal care. As such females should have developed reliable, condition-dependent criteria to choose mates to guarantee survival and care for offspring. We show that females actively chose males experienced in fatherhood over others. In the absence of experienced males dominance, as evident from male-male fights, is utilized for mate selection. The preference for experienced males is not affected by previous social interactions between the individuals. We show that the choice of the partner is based on chemical signals demonstrating a ‘scent of experience’ to females providing evidence for the role of chemical signals in sexual selection for paternal care adding to our understanding of the mechanisms regulating condition-dependent mate choice.

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

2009-11-04T08:00:00Z

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.

Why Amphibians Are More Sensitive than Mammals to Xenobiotics

2009-11-04T08:00:00Z

Dramatic declines in amphibian populations have been described all over the world since the 1980s. The evidence that the sensitivity to environmental threats is greater in amphibians than in mammals has been generally linked to the observation that amphibians are characterized by a rather permeable skin. Nevertheless, a numerical comparison of data of percutaneous (through the skin) passage between amphibians and mammals is lacking. Therefore, in this investigation we have measured the percutaneous passage of two test molecules (mannitol and antipyrine) and three heavily used herbicides (atrazine, paraquat and glyphosate) in the skin of the frog Rana esculenta (amphibians) and of the pig ear (mammals), by using the same experimental protocol and a simple apparatus which minimizes the edge effect, occurring when the tissue is clamped in the usually used experimental device.

The percutaneous passage (P) of each substance is much greater in frog than in pig. LogP is linearly related to logK_ow (logarithm of the octanol-water partition coefficient). The measured P value of atrazine was about 134 times larger than that of glyphosate in frog skin, but only 12 times in pig ear skin. The FoD value (P_frog/P_pig) was 302 for atrazine, 120 for antipyrine, 66 for mannitol, 29 for paraquat, and 26 for glyphosate.

The differences in structure and composition of the skin between amphibians and mammals are discussed.

Effects of 15-Deoxy-Δ^12,14-Prostaglandin J2 (15d-PGJ2) and Rosiglitazone on Human Vδ2⁺ T Cells

2009-11-04T08:00:00Z

Background

Thiazolidinediones (TZD) class of drugs, and 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) are immune regulators predicted to modulate human autoimmune disease. Their effects on γδ T cells, which are involved in animal model and human and animal autoimmune diseases, are unknown.

Methodology/Principal Findings

We characterized the activity of rosiglitazone (from the TZD class of drugs) and 15d-PGJ2 in human Vδ2 T cells. We found that 15d-PGJ2 and rosiglitazone had different effects on Vδ2 T cell functions. Both 15d-PGJ2 and rosiglitazone suppressed Vδ2 T cell proliferation in response to IPP and IL2. However, only 15d-PGJ2 suppressed functional responses including cytokine production, degranulation and cytotoxicity against tumor cells. The mechanism for 15d-PGJ2 effects on Vδ2 T cells acts through inhibiting Erk activation. In contrast, rosiglitazone did not affect Erk activation but the IL2 signaling pathway, which accounts for rosiglitazone suppression of IL2-dependent, Vδ2 T cell proliferation without affecting TCR-dependent functions. Rosiglitazone and 15d-PGJ2 are designed to be peroxisome proliferator-activated receptor gamma (PPARγ) ligands and PPARγ was expressed in Vδ2 T cell. Surprisingly, when PPARγ levels were lowered by specific siRNA, 15d-PGJ2 and rosiglitazone were still active, suggesting their target of action induces cellular proteins other than PPARγ.

Conclusions/Significance

The current findings expand our understanding of how the immune system is regulated by rosiglitazone and 15d-PGJ2 and will be important to evaluate these compounds as therapeutic agents in human autoimmune disease.

A Concerted Kinase Interplay Identifies PPARγ as a Molecular Target of Ghrelin Signaling in Macrophages

2009-11-04T08:00:00Z

The peroxisome proliferator-activator receptor PPARγ plays an essential role in vascular biology, modulating macrophage function and atherosclerosis progression. Recently, we have described the beneficial effect of combined activation of the ghrelin/GHS-R1a receptor and the scavenger receptor CD36 to induce macrophage cholesterol release through transcriptional activation of PPARγ. Although the interplay between CD36 and PPARγ in atherogenesis is well recognized, the contribution of the ghrelin receptor to regulate PPARγ remains unknown. Here, we demonstrate that ghrelin triggers PPARγ activation through a concerted signaling cascade involving Erk1/2 and Akt kinases, resulting in enhanced expression of downstream effectors LXRα and ABC sterol transporters in human macrophages. These effects were associated with enhanced PPARγ phosphorylation independently of the inhibitory conserved serine-84. Src tyrosine kinase Fyn was identified as being recruited to GHS-R1a in response to ghrelin, but failure of activated Fyn to enhance PPARγ Ser-84 specific phosphorylation relied on the concomitant recruitment of docking protein Dok-1, which prevented optimal activation of the Erk1/2 pathway. Also, substitution of Ser-84 preserved the ghrelin-induced PPARγ activity and responsiveness to Src inhibition, supporting a mechanism independent of Ser-84 in PPARγ response to ghrelin. Consistent with this, we found that ghrelin promoted the PI3-K/Akt pathway in a Gα_q-dependent manner, resulting in Akt recruitment to PPARγ, enhanced PPARγ phosphorylation and activation independently of Ser-84, and increased expression of LXRα and ABCA1/G1. Collectively, these results illustrate a complex interplay involving Fyn/Dok-1/Erk and Gα_q/PI3-K/Akt pathways to transduce in a concerted manner responsiveness of PPARγ to ghrelin in macrophages.

The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

2009-11-04T08:00:00Z

Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.

Brain Stem Death as the Vital Determinant for Resumption of Spontaneous Circulation after Cardiac Arrest in Rats

2009-11-04T08:00:00Z

Background

Spontaneous circulation returns to less than half of adult cardiac arrest victims who received in-hospital resuscitation. One clue for this disheartening outcome arises from the prognosis that asystole invariably takes place, after a time lag, on diagnosis of brain stem death. The designation of brain stem death as the point of no return further suggests that permanent impairment of the brain stem cardiovascular regulatory machinery precedes death. It follows that a crucial determinant for successful revival of an arrested heart is that spontaneous circulation must resume before brain stem death commences. Here, we evaluated the hypothesis that maintained functional integrity of the rostral ventrolateral medulla (RVLM), a neural substrate that is intimately related to brain stem death and central circulatory regulation, holds the key to the vital time-window between cardiac arrest and resumption of spontaneous circulation.

Methodology/Principal Findings

An animal model of brain stem death employing the pesticide mevinphos as the experimental insult in Sprague-Dawley rats was used. Intravenous administration of lethal doses of mevinphos elicited an abrupt cardiac arrest, accompanied by elevated systemic arterial pressure and anoxia, augmented neuronal excitability and enhanced microvascular perfusion in RVLM. This period represents the vital time-window between cardiac arrest and resumption of spontaneous circulation in our experimental model. Animals with restored spontaneous circulation exhibited maintained neuronal functionality in RVLM beyond this critical time-window, alongside resumption of baseline tissue oxygen and enhancement of local blood flow. Intriguingly, animals that subsequently died manifested sustained anoxia, diminished local blood flow, depressed mitochondrial electron transport activities and reduced ATP production, leading to necrotic cell death in RVLM. That amelioration of mitochondrial dysfunction and bioenergetic failure in RVLM by coenzyme Q10, the mobile electron carrier in mitochondrial respiratory chain, or oxygenation restored spontaneous circulation further established a causal relationship between functionality of RVLM and resumed spontaneous circulation after cardiac arrest.

Conclusions/Significance

We conclude that whereas necrotic cell death because of bioenergetic failure triggered by anoxia in RVLM, which precipitates brain stem death, negates resuscitation of an arrested heart, maintained functional integrity of this neural substrate holds the key to resumption of spontaneous circulation after cardiac arrest in rats.

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

2009-11-04T08:00:00Z

Background

Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.

Methodology/Principal Findings

Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect ≤1% mutant cells, provided that samples yielded cycle thresholds (Ct) <29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%).

Conclusions/Significance

Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

Chimerism in Wild Adult Populations of the Broadcast Spawning Coral Acropora millepora on the Great Barrier Reef

2009-11-04T08:00:00Z

Background

Chimeras are organisms containing tissues or cells of two or more genetically distinct individuals, and are known to exist in at least nine phyla of protists, plants, and animals. Although widespread and common in marine invertebrates, the extent of chimerism in wild populations of reef corals is unknown.

Methodology/Principal Findings

The extent of chimerism was explored within two populations of a common coral, Acropora millepora, on the Great Barrier Reef, Australia, by using up to 12 polymorphic DNA microsatellite loci. At least 2% and 5% of Magnetic Island and Pelorus Island populations of A. millepora, respectively, were found to be chimeras (3% overall), based on conservative estimates. A slightly less conservative estimate indicated that 5% of colonies in each population were chimeras. These values are likely to be vast underestimates of the true extent of chimerism, as our sampling protocol was restricted to a maximum of eight branches per colony, while most colonies consist of hundreds of branches. Genotypes within chimeric corals showed high relatedness, indicating that genetic similarity is a prerequisite for long-term acceptance of non-self genotypes within coral colonies.

Conclusions/Significance

While some brooding corals have been shown to form genetic chimeras in their early life history stages under experimental conditions, this study provides the first genetic evidence of the occurrence of coral chimeras in the wild and of chimerism in a broadcast spawning species. We hypothesize that chimerism is more widespread in corals than previously thought, and suggest that this has important implications for their resilience, potentially enhancing their capacity to compete for space and respond to stressors such as pathogen infection.

Filamentous Biopolymers on Surfaces: Atomic Force Microscopy Images Compared with Brownian Dynamics Simulation of Filament Deposition

2009-11-04T08:00:00Z

Nanomechanical properties of filamentous biopolymers, such as the persistence length, may be determined from two-dimensional images of molecules immobilized on surfaces. For a single filament in solution, two principal adsorption scenarios are possible. Both scenarios depend primarly on the interaction strength between the filament and the support: i) For interactions in the range of the thermal energy, the filament can freely equilibrate on the surface during adsorption; ii) For interactions much stronger than the thermal energy, the filament will be captured by the surface without having equilibrated. Such a ‘trapping’ mechanism leads to more condensed filament images and hence to a smaller value for the apparent persistence length. To understand the capture mechanism in more detail we have performed Brownian dynamics simulations of relatively short filaments by taking the two extreme scenarios into account. We then compared these ‘ideal’ adsorption scenarios with observed images of immobilized vimentin intermediate filaments on different surfaces. We found a good agreement between the contours of the deposited vimentin filaments on mica (‘ideal’ trapping) and on glass (‘ideal’ equilibrated) with our simulations. Based on these data, we have developed a strategy to reliably extract the persistence length of short worm-like chain fragments or network forming filaments with unknown polymer-surface interactions.

In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

2009-11-03T08:00:00Z

Background

The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro.

Methodology

Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture.

Principal Findings

The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line.

Conclusions

The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

2009-11-03T08:00:00Z

Background

While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).

Methods/Principal Findings

Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.

Conclusions

This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

The Vitamin B1 Metabolism of Staphylococcus aureus Is Controlled at Enzymatic and Transcriptional Levels

2009-11-03T08:00:00Z

Vitamin B1 is in its active form thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and thiamine phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the thiamine pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels.

Monocyte Chemotactic Protein-1 (MCP-1) and Growth Factors Called into Question as Markers of Prolonged Psychosocial Stress

2009-11-03T08:00:00Z

Background

Psychosocial stress is becoming a major contributor to increased mental ill-health and sick leave in many countries. Valid markers of chronic stress would be valuable for diagnostic and prognostic purposes. A recent study suggested monocyte chemotactic protein-1 (MCP-1), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as markers of chronic stress. We aimed to confirm these potential biomarkers of prolonged psychosocial stress in female patients.

Methodology/Principal Findings

Circulating levels of MCP-1, EGF and VEGF, along with several other cytokines, were measured in plasma from 42 female patients suffering from exhaustion due to prolonged psychosocial stress and 42 control subjects, using a protein biochip immunoassay. There were no significant differences between patients and controls in any of the cytokines or growth factors analyzed. Furthermore, when using a different protein bioassay and reanalyzing MCP-1 and VEGF in the same samples, markedly different levels were obtained. To further explore if inflammation is present in patients with exhaustion, the classical inflammatory marker C-reactive protein (CRP) was measured in another group of patients (n = 89) and controls (n = 88) showing a small but significant increase of CRP levels in the patients.

Conclusions/Significance

MCP-1, EGF and VEGF may not be suitable markers of prolonged psychosocial stress as previously suggested. Furthermore, significant differences were obtained when using two different protein assays measuring the same samples, indicating that comparing studies where different analytic techniques have been used might be difficult. Increased levels of CRP indicate that low-grade inflammation might be present in patients with exhaustion due to prolonged stress exposure but this inflammation does not seem to be reflected by increase in circulating MCP-1 or other cytokines measured.

Differential Deployment of REST and CoREST Promotes Glial Subtype Specification and Oligodendrocyte Lineage Maturation

2009-11-03T08:00:00Z

Background

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master transcriptional regulator that binds to numerous genomic RE1 sites where it acts as a molecular scaffold for dynamic recruitment of modulatory and epigenetic cofactors, including corepressor for element-1-silencing transcription factor (CoREST). CoREST also acts as a hub for various cofactors that play important roles in epigenetic remodeling and transcriptional regulation. While REST can recruit CoREST to its macromolecular complex, CoREST complexes also function at genomic sites independently of REST. REST and CoREST perform a broad array of context-specific functions, which include repression of neuronal differentiation genes in neural stem cells (NSCs) and other non-neuronal cells as well as promotion of neurogenesis. Despite their involvement in multiple aspects of neuronal development, REST and CoREST are not believed to have any direct modulatory roles in glial cell maturation.

Methodology/Principal Findings

We challenged this view by performing the first study of REST and CoREST in NSC-mediated glial lineage specification and differentiation. Utilizing ChIP on chip (ChIP-chip) assays, we identified distinct but overlapping developmental stage-specific profiles for REST and CoREST target genes during astrocyte (AS) and oligodendrocyte (OL) lineage specification and OL lineage maturation and myelination, including many genes not previously implicated in glial cell biology or linked to REST and CoREST regulation. Amongst these factors are those implicated in macroglial (AS and OL) cell identity, maturation, and maintenance, such as members of key developmental signaling pathways and combinatorial transcription factor codes.

Conclusions/Significance

Our results imply that REST and CoREST modulate not only neuronal but also glial lineage elaboration. These factors may therefore mediate critical developmental processes including the coupling of neurogenesis and gliogenesis and neuronal-glial interactions that underlie synaptic and neural network plasticity and homeostasis in health and in specific neurological disease states.

Cytokine Levels Correlate with Immune Cell Infiltration after Anti-VEGF Therapy in Preclinical Mouse Models of Breast Cancer

2009-11-03T08:00:00Z

The effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. However, no studies have compared the effectiveness of mechanistically different anti-VEGF inhibitors with respect to changes in tumor growth and alterations in the tumor microenvironment. In this study we use three distinct breast cancer models, a MDA-MB-231 xenograft model, a 4T1 syngenic model, and a transgenic model using MMTV-PyMT mice, to explore the effects of various anti-VEGF therapies on tumor vasculature, immune cell infiltration, and cytokine levels. Tumor vasculature and immune cell infiltration were evaluated using immunohistochemistry. Cytokine levels were evaluated using ELISA and electrochemiluminescence. We found that blocking the activation of VEGF receptor resulted in changes in intra-tumoral cytokine levels, specifically IL-1β, IL-6 and CXCL1. Modulation of the level these cytokines is important for controlling immune cell infiltration and ultimately tumor growth. Furthermore, we demonstrate that selective inhibition of VEGF binding to VEGFR2 with r84 is more effective at controlling tumor growth and inhibiting the infiltration of suppressive immune cells (MDSC, Treg, macrophages) while increasing the mature dendritic cell fraction than other anti-VEGF strategies. In addition, we found that changes in serum IL-1β and IL-6 levels correlated with response to therapy, identifying two possible biomarkers for assessing the effectiveness of anti-VEGF therapy in breast cancer patients.

Evolutionary History of the HAP2/GCS1 Gene and Sexual Reproduction in Metazoans

2009-11-03T08:00:00Z

The HAP2/GCS1 gene first appeared in the common ancestor of plants, animals, and protists, and is required in the male gamete for fusion to the female gamete in the unicellular organisms Chlamydomonas and Plasmodium. We have identified a HAP2/GCS1 gene in the genome sequence of the sponge Amphimedon queenslandica. This finding provides a continuous evolutionary history of HAP2/GCS1 from unicellular organisms into the metazoan lineage. Divergent versions of the HAP2/GCS1 gene are also present in the genomes of some but not all arthropods. By examining the expression of the HAP2/GCS1 gene in the cnidarian Hydra, we have found the first evidence supporting the hypothesis that HAP2/GCS1 was used for male gamete fusion in the ancestor of extant metazoans and that it retains that function in modern cnidarians.

Mbd3, a Component of NuRD/Mi-2 Complex, Helps Maintain Pluripotency of Mouse Embryonic Stem Cells by Repressing Trophectoderm Differentiation

2009-11-03T08:00:00Z

Embryonic stem cells (ES cells) can differentiate into cells derived from all three germ layers and extraembryonic tissues. While transcription factors such as, Oct4 and Nanog are well known for their requirements for undifferentiated ES cell growth, mechanisms of epigenetic repression of germ layer specific differentiation in ES cells are not well understood. Here, we investigate functions of Mbd3, a component of nucleosome remodeling and histone deacetylation complex (NuRD/Mi-2) in mouse ES cells. We find that compared to wild type ES cells, Mbd3 knockdown cells show elevated RNA expression of trophectoderm markers, including Cdx2, Eomesodermin, and Hand1. In parallel, these cells show an increased acetylation level of histone 3 in promoters of the respective genes, suggesting Mbd3 plays a role in repression of these genes in undifferentiated ES cells. However, these changes are not sufficient for definitive differentiation to trophectoderm (TE) in chimeric embryos. When further cultured in ES medium without LIF or in trophoblast stem (TS) cell medium, Mbd3 knockdown cells differentiate into TE cells, which express Cdx2 and, at later stages, trophoblast lineage specific marker Cadherin 3. These results suggest that Mbd3 helps restrict ES cells from differentiating towards the trophectoderm lineage and is an important epigenetic player in maintaining full pluripotency of mouse ES cells.

A Protein-Protein Interaction Map of the Trypanosoma brucei Paraflagellar Rod

2009-11-03T08:00:00Z

We have conducted a protein interaction study of components within a specific sub-compartment of a eukaryotic flagellum. The trypanosome flagellum contains a para-crystalline extra-axonemal structure termed the paraflagellar rod (PFR) with around forty identified components. We have used a Gateway cloning approach coupled with yeast two-hybrid, RNAi and 2D DiGE to define a protein-protein interaction network taking place in this structure. We define two clusters of interactions; the first being characterised by two proteins with a shared domain which is not sufficient for maintaining the interaction. The other cohort is populated by eight proteins, a number of which possess a PFR domain and sub-populations of this network exhibit dependency relationships. Finally, we provide clues as to the structural organisation of the PFR at the molecular level. This multi-strand approach shows that protein interactome data can be generated for insoluble protein complexes.

Fyn Mediates Leptin Actions in the Thymus of Rodents

2009-11-03T08:00:00Z

Background

Several effects of leptin in the immune system rely on its capacity to modulate cytokine expression and apoptosis in the thymus. Surprisingly, some of these effects are dependent on signal transduction through the IRS1/PI3-kinase, but not on the activation of JAK2. Since all the well known effects of leptin in different cell types and tissues seem to be dependent on JAK2 activation, we hypothesized that, at least for the control of thymic function, another, unknown kinase could mediate the transduction of the leptin signal from the ObR towards the IRS1/PI3-kinase signaling cascade.

Methodology/Principal Findings

Here, by employing immunoblot, real-time PCR and flow citometry we show that the tyrosine kinase, Fyn, is constitutively associated with the ObR in thymic cells. Following a leptin stimulus, Fyn undergoes an activating tyrosine phosphorylation and a transient association with IRS1. All these effects are independent of JAK2 activation and, upon Fyn inhibition, the signal transduction towards IRS1/PI3-kinase is abolished. In addition, the inhibition of Fyn significantly modifies the effects of leptin on thymic cytokine expression.

Conclusion/Significance

Therefore, in the thymus, Fyn acts as a tyrosine kinase that transduces the leptin signal independently of JAK2 activation, and mediates some of the immunomodulatory effects of leptin in this tissue.

A Non-Redundant Role for Drosophila Mkk4 and Hemipterous/Mkk7 in TAK1-Mediated Activation of JNK

2009-11-03T08:00:00Z

Background

The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1.

Methodology/Principal Findings

In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation.

Conclusions/Significance

In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.

Somatosensory Cortices Are Required for the Acquisition of Morphine-Induced Conditioned Place Preference

2009-11-03T08:00:00Z

Background

Sensory system information is thought to play an important role in drug addiction related responses. However, how somatic sensory information participates in the drug related behaviors is still unclear. Many studies demonstrated that drug addiction represents a pathological usurpation of neural mechanisms of learning and memory that normally relate to the pursuit of rewards. Thus, elucidate the role of somatic sensory in drug related learning and memory is of particular importance to understand the neurobiological mechanisms of drug addiction.

Principal Findings

In the present study, we investigated the role of somatosensory system in reward-related associative learning using the conditioned place preference model. Lesions were made in somatosensory cortices either before or after conditioning training. We found that lesion of somatosensory cortices before, rather than after morphine conditioning impaired the acquisition of place preference.

Conclusion

These results demonstrate that somatosensory cortices are necessary for the acquisition but not retention of morphine induced place preference.

An Integrated Analysis of Molecular Acclimation to High Light in the Marine Diatom Phaeodactylum tricornutum

2009-11-03T08:00:00Z

Photosynthetic diatoms are exposed to rapid and unpredictable changes in irradiance and spectral quality, and must be able to acclimate their light harvesting systems to varying light conditions. Molecular mechanisms behind light acclimation in diatoms are largely unknown. We set out to investigate the mechanisms of high light acclimation in Phaeodactylum tricornutum using an integrated approach involving global transcriptional profiling, metabolite profiling and variable fluorescence technique. Algae cultures were acclimated to low light (LL), after which the cultures were transferred to high light (HL). Molecular, metabolic and physiological responses were studied at time points 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h after transfer to HL conditions. The integrated results indicate that the acclimation mechanisms in diatoms can be divided into an initial response phase (0–0.5 h), an intermediate acclimation phase (3–12 h) and a late acclimation phase (12–48 h). The initial phase is recognized by strong and rapid regulation of genes encoding proteins involved in photosynthesis, pigment metabolism and reactive oxygen species (ROS) scavenging systems. A significant increase in light protecting metabolites occur together with the induction of transcriptional processes involved in protection of cellular structures at this early phase. During the following phases, the metabolite profiling display a pronounced decrease in light harvesting pigments, whereas the variable fluorescence measurements show that the photosynthetic capacity increases strongly during the late acclimation phase. We show that P. tricornutum is capable of swift and efficient execution of photoprotective mechanisms, followed by changes in the composition of the photosynthetic machinery that enable the diatoms to utilize the excess energy available in HL. Central molecular players in light protection and acclimation to high irradiance have been identified.

Male Perspectives on Incorporating Men into Antenatal HIV Counseling and Testing

2009-11-02T08:00:00Z

Background

Male partner involvement in antenatal voluntary HIV counseling and testing (VCT) has been shown to increase uptake of interventions to reduce the risk of HIV transmission in resource-limited settings. We aimed to identify methods for increasing male involvement in antenatal VCT and determine male correlates of accepting couple counseling in these settings.

Methodology/Principal Findings

We invited women presenting to a Nairobi antenatal clinic to return with their male partners for individual or couples VCT. Male attitudes towards VCT and correlates of accompanying female partners to antenatal clinic and receiving couple counseling were determined. Of 1,993 women who invited their partner, 313 (16%) returned with their partners to ANC. Men attending antenatal clinic were married (>99%), employed (98%), and unlikely to report prior HIV testing (14%). Wanting an HIV test (87%) or health information (11%) were the most commonly cited reasons for attending. Most (95%) men who came to antenatal clinic accepted HIV testing and 39% elected to receive counseling as a couple. Men who received counseling with partners were younger, had fewer children, and were less knowledgeable about prevention of mother-to-child HIV transmission (PMTCT) than those who received counseling individually (p<0.05). Only 27% of men stated they would prefer HIV testing at a site other than the ANC. There was agreement between male and female reports for sociodemographic characteristics; however, men were more likely to report HIV preventive behaviors and health communication within the partnership than their partners (p<0.05).

Conclusions/Significance

Offering VCT services to men at antenatal clinic with options for couple and individual counseling is an important opportunity and acceptable strategy for increasing male involvement in PMTCT and promoting male HIV testing.

Repertoires of the Nucleosome-Positioning Dinucleotides

2009-11-02T08:00:00Z

It is generally accepted that the organization of eukaryotic DNA into chromatin is strongly governed by a code inherent in the genomic DNA sequence. This code, as well as other codes, is superposed on the triplets coding for amino acids. The history of the chromatin code started three decades ago with the discovery of the periodic appearance of certain dinucleotides, with AA/TT and RR/YY giving the strongest signals, all with a period of 10.4 bases. Every base-pair stack in the DNA duplex has specific deformation properties, thus favoring DNA bending in a specific direction. The appearance of the corresponding dinucleotide at the distance 10.4 xn bases will facilitate DNA bending in that direction, which corresponds to the minimum energy of DNA folding in the nucleosome. We have analyzed the periodic appearances of all 16 dinucleotides in the genomes of thirteen different eukaryotic organisms. Our data show that a large variety of dinucleotides (if not all) are, apparently, contributing to the nucleosome positioning code. The choice of the periodical dinucleotides differs considerably from one organism to another. Among other 10.4 base periodicities, a strong and very regular 10.4 base signal was observed for CG dinucleotides in the genome of the honey bee A. mellifera. Also, the dinucleotide CG appears as the only periodical component in the human genome. This observation seems especially relevant since CpG methylation is well known to modulate chromatin packing and regularity. Thus, the selection of the dinucleotides contributing to the chromatin code is species specific, and may differ from region to region, depending on the sequence context.

A Genome-Wide Analysis of Small Regulatory RNAs in the Human Pathogen Group A Streptococcus

2009-11-02T08:00:00Z

The coordinated regulation of gene expression is essential for pathogens to infect and cause disease. A recently appreciated mechanism of regulation is that afforded by small regulatory RNA (sRNA) molecules. Here, we set out to assess the prevalence of sRNAs in the human bacterial pathogen group A Streptococcus (GAS). Genome-wide identification of candidate GAS sRNAs was performed through a tiling Affymetrix microarray approach and identified 40 candidate sRNAs within the M1T1 GAS strain MGAS2221. Together with a previous bioinformatic approach this brings the number of novel candidate sRNAs in GAS to 75, a number that approximates the number of GAS transcription factors. Transcripts were confirmed by Northern blot analysis for 16 of 32 candidate sRNAs tested, and the abundance of several of these sRNAs were shown to be temporally regulated. Six sRNAs were selected for further study and the promoter, transcriptional start site, and Rho-independent terminator identified for each. Significant variation was observed between the six sRNAs with respect to their stability during growth, and with respect to their inter- and/or intra-serotype-specific levels of abundance. To start to assess the contribution of sRNAs to gene regulation in M1T1 GAS we deleted the previously described sRNA PEL from four clinical isolates. Data from genome-wide expression microarray, quantitative RT-PCR, and Western blot analyses are consistent with PEL having no regulatory function in M1T1 GAS. The finding that candidate sRNA molecules are prevalent throughout the GAS genome provides significant impetus to the study of this fundamental gene-regulatory mechanism in an important human pathogen.

Studies of α-Helicity and Intersegmental Interactions in Voltage-Gated Na⁺ Channels: S2D4

2009-11-02T08:00:00Z

Much data, including crystallographic, support structural models of sodium and potassium channels consisting of S1–S4 transmembrane segments (the “voltage-sensing domain”) clustered around a central pore-forming region (S5–S6 segments and the intervening loop). Voltage gated sodium channels have four non-identical domains which differentiates them from the homotetrameric potassium channels that form the basis for current structural models. Since potassium and sodium channels also exhibit many different functional characteristics and the fourth domain (D4) of sodium channels differs in function from other domains (D1–D3), we have explored its structure in order to determine whether segments in D4 of sodium channels differ significantly from that determined for potassium channels. We have probed the secondary and tertiary structure and the role of the individual amino acid residues of the S2D4) of Na_v1.4 by employing cysteine-scanning mutagenesis (with tryptophan and glutamine substituted for native cysteine). A Fourier transform power spectrum of perturbations in free energy of steady-state inactivation gating (using midpoint potentials and slopes of Boltzmann equation fits of channel availability, h_∞-V plots) indicates a substantial amount of α-helical structure in S2D4 (peak at 106°, α-Periodicity Index (α-PI) of 3.10), This conclusion is supported by α-PI values of 3.28 and 2.84 for the perturbations in rate constants of entry into (β) and exit from (α) fast inactivation at 0 mV for mutant channels relative to WT channels assuming a simple two-state model for transition from the open to inactivated state. The results of cysteine substitution at the two most sensitive sites of the S2D4 α-helix (N1382 and E1392C) support the existence of electrostatic network interactions between S2 and other transmembrane segments within Na_v1.4D4 similar to but not identical to those proposed for K⁺ channels.

Relationship between CAD Risk Genotype in the Chromosome 9p21 Locus and Gene Expression. Identification of Eight New ANRIL Splice Variants

2009-11-02T08:00:00Z

Background

Several genome-wide association studies have recently linked a group of single nucleotide polymorphisms in the 9p21 region with cardiovascular disease. The molecular mechanisms of this link are not fully understood. We investigated five different expression microarray datasets in order to determine if the genotype had effect on expression of any gene transcript in aorta, mammary artery, carotid plaque and lymphoblastoid cells.

Methodology/Principal Findings

After multiple testing correction, no genes were found to have relation to the rs2891168 risk genotype, either on a genome-wide scale or on a regional (8 MB) scale. The neighbouring ANRIL gene was found to have eight novel transcript variants not previously known from literature and these varied by tissue type. We therefore performed a detailed probe-level analysis and found small stretches of significant relation to genotype but no consistent associations. In all investigated tissues we found an inverse correlation between ANRIL and the MTAP gene and a positive correlation between ANRIL and CDKN2A and CDKN2B.

Conclusions/Significance

Investigation of relation of the risk genotype to gene expression is complicated by the transcript complexity of the locus. With our investigation of a range of relevant tissue we wish to underscore the need for careful attention to the complexity of the alternative splicing issues in the region and its implications to the design of future gene expression studies.

Acceptability of Male Circumcision for the Prevention of HIV/AIDS in the Dominican Republic

2009-11-02T08:00:00Z

Background

Male circumcision (MC) is an effective strategy to prevent HIV infection in heterosexual men. To our knowledge, there are no studies of the acceptability of this procedure in the Dominican Republic (DR). The main objective of this study was to assess the acceptability of MC to prevent HIV transmission among men ages 18 to 50 years in the Altagracia Province in the Dominican Republic. Because differences in culture and beliefs between Haitians and Dominicans could potentially influence their acceptability of MC, we conducted a comparative analysis based on national origin.

Methods

A survey was administered to a convenience sample of 368 men. The questionnaire was divided in 3 sections: 1) Background demographics (including national origin), 2) Male circumcision and 3) Sexual health. Stratified and logistic multivariate regression analyses were performed to identify factors associated with the acceptability of MC.

Results

The sample consisted of 238 (65%) Dominicans and 130 (35%) Haitian immigrants. Almost all participants were uncircumcised (95%) and about half (52%) were single. The overall acceptability of MC was 29%. The number of men willing to be circumcised increased to 67% after an information session explaining the benefits of the procedure. 74% of men reported that they would be willing to circumcise their sons after hearing that information. In multivariate analysis, Haitian nationality (OR = 1.86, 95% CI 1.01–3.41), knowing that circumcision improves hygiene (OR = 2.78, 95% CI 1.29–6.0) and not believing that circumcision decreases sexual pleasure (OR = 2.18, 95% CI 1.20–3.94) were associated with a higher acceptability of the procedure. Although age was not significantly associated with the willingness to be circumcised in the multivariate analysis, stratified analysis based on national origin suggested that younger Dominicans (<30 years of age) are more likely to accept the procedure when compared to their older counterparts (OR = 2.17, 95% CI 1.14–4.12).

Conclusions

An important number of sexually active men in the DR may be willing to be circumcised if educational resources detailing the benefits of the procedure are made available. These educational activities would constitute a great opportunity to teach about sexual health and reinforce safe sex practices.

Expression Profile of MicroRNAs in Young Stroke Patients

2009-11-02T08:00:00Z

Background

The methods currently available for diagnosis and prognosis of cerebral ischaemia still require further improvements. Micro-RNAs (small non-coding RNAs) have been recently reported as useful biomarkers in diseases such as cancer and diabetes. We therefore carried out microRNA (miRNA) profiling from peripheral blood to detect and identify characteristic patterns in ischaemic stroke.

Methods/Principal Findings

The ischaemic stroke patients aged between 18–49 years, characterized based on World Health Organization clinical criteria were further classified according to TOAST classification, a) Large-vessel atherosclerosis [n = 8] b) Small-vessel disease [n = 3] c) Cardioembolism [n = 5] d) Undetermined cause [n = 3]. The patients' functional status at the time of blood sampling (at the outpatient clinics) was evaluated with the modified Rankin Scale (mRS). Blood samples from normal (n = 5) individuals were used as controls. Total RNA extracted from whole blood was subjected to miroRNA profiling and real-time PCR analysis.

miRNAs that are implicated in the endothelial/vascular function, erythropoiesis, angiogenesis and neural function showed differential expression profile as compared to the normal control. Interestingly, miRNAs that are involved in hypoxic conditions have also been found in our miRNA profiles.

Conclusion

We demonstrate that the peripheral blood miRNAs and their profiles can be developed as biomarkers in diagnosis and prognosis of cerebral ischaemic stroke. The dysregulated miRNAs have been detectable even after several months from the onset of stroke in what is usually regarded as neurologically stable patients.

The Contribution of Family Planning towards the Prevention of Vertical HIV Transmission in Uganda

2009-11-02T08:00:00Z

Background

Uganda has one of the highest total fertility rates (TFR) worldwide. We compared the effects of antiretroviral (ARV) prophylaxis for the prevention of mother-to-child HIV transmission (PMTCT) to that of existing family planning (FP) use and estimated the burden of pediatric HIV disease due to unwanted fertility.

Methodology/Principal Findings

Using the demographic software Spectrum, a baseline mathematical projection to estimate the current pediatric HIV burden in Uganda was compared to three hypothetical projections: 1) without ARV-PMTCT (to estimate the effect of ARV-PMTCT), 2) without contraception (effect of existing FP use), 3) without unwanted fertility (effect of unmet FP needs). Key input parameters included HIV prevalence, ARV-PMTCT uptake, MTCT probabilities, and TFR. We estimate that in 2007, an estimated 25,000 vertical infections and 17,000 pediatric AIDS deaths occurred (baseline projection). Existing ARV-PMTCT likely averted 8.1% of infections and 8.5% of deaths. FP use likely averted 19.7% of infections and 13.1% of deaths. Unwanted fertility accounted for 21.3% of infections and 13.4% of deaths. During 2008–2012, an estimated 131,000 vertical infections and 71,000 pediatric AIDS deaths will occur. The projected scale up of ARV-PMTCT (from 39%–57%) may avert 18.1% of infections and 24.5% of deaths. Projected FP use may avert 21.6% of infections and 18.5% of deaths. Unwanted fertility will account for 24.5% of infections and 19.8% of deaths.

Conclusions

Existing FP use contributes as much or more than ARV-PMTCT in mitigating pediatric HIV in Uganda. Expanding FP services can substantially contribute towards PMTCT.

Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

2009-11-02T08:00:00Z

Background

There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Δ32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation.

Methods and Findings

We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV)/HIV-1 (SHIV) chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT) transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Student's t-test), but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly translated to the clinic.

Conclusions

Here we demonstrate the ability to select protected HSC-derived lymphocytes in vivo in a clinically relevant nonhuman primate model of HIV/SHIV infection. This approach can now be evaluated in human clinical trials in AIDS lymphoma patients. In this patient setting, chemotherapy would not only kill malignant cells, but would also increase the number of MGMTP140K-expressing HIV-resistant cells. This approach should allow for high levels of HIV-protected cells in AIDS patients to evaluate AIDS gene therapy.

Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

2009-11-02T08:00:00Z

Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism.

Bub3 Is a Spindle Assembly Checkpoint Protein Regulating Chromosome Segregation during Mouse Oocyte Meiosis

2009-11-02T08:00:00Z

In mitosis, the spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad)1–3, budding uninhibited by benzimidazole (Bub)1, Bub3, and monopolar spindle 1(Mps1). During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.

Transcriptional Effects of Glucocorticoid Receptors in the Dentate Gyrus Increase Anxiety-Related Behaviors

2009-11-02T08:00:00Z

The Glucocorticoid Receptor (GR) is a transcription factor ubiquitously expressed in the brain. Activation of brain GRs by high levels of glucocorticoid (GC) hormones modifies a large variety of physiological and pathological-related behaviors. Unfortunately the specific cellular targets of GR-mediated behavioral effects of GC are still largely unknown. To address this issue, we generated a mutated form of the GR called ΔGR. ΔGR is a constitutively transcriptionally active form of the GR that is localized in the nuclei and activates transcription without binding to glucocorticoids. Using the tetracycline-regulated system (Tet-OFF), we developed an inducible transgenic approach that allows the expression of the ΔGR in specific brain areas. We focused our study on a mouse line that expressed ΔGR almost selectively in the glutamatergic neurons of the dentate gyrus (DG) of the hippocampus. This restricted expression of the ΔGR increased anxiety-related behaviors without affecting other behaviors that could indirectly influence performance in anxiety-related tests. This behavioral phenotype was also associated with an up-regulation of the MAPK signaling pathway and Egr-1 protein in the DG. These findings identify glutamatergic neurons in the DG as one of the cellular substrate of stress-related pathologies.

AAV Recombineering with Single Strand Oligonucleotides

2009-11-02T08:00:00Z

Adeno-associated virus (AAV) transduction initiates a signaling cascade that culminates in a transient DNA damage response. During this time, host DNA repair proteins convert the linear single-strand AAV genomes to double-strand circular monomers and concatemers in processes stimulated by the AAV inverted terminal repeats (ITRs). As the orientation of AAV genome concatemerization appears unbiased, the likelihood of concatemerization in a desired orientation is low (less than 1 in 6). Using a novel recombineering method, Oligo-Assisted AAV Genome Recombination (OAGR), this work demonstrates the ability to direct concatemerization specifically to a desired orientation in human cells. This was achieved by a single-strand DNA oligonucleotide (oligo) displaying homology to distinct AAV genomes capable of forming an intermolecular bridge for recombination. This DNA repair process results in concatemers with genomic junctions corresponding to the sequence of oligo homology. Furthermore, OAGR was restricted to single-strand, not duplexed, AAV genomes suggestive of replication-dependent recombination. Consistent with this process, OAGR demonstrated oligo polarity biases in all tested configurations except when a portion of the oligo targeted the ITR. This approach, in addition to being useful for the elucidation of intermolecular homologous recombination, may find eventual relevance for AAV mediated large gene therapy.

Heat Shock Protein-Derived T-Cell Epitopes Contribute to Autoimmune Inflammation in Pediatric Crohn's Disease

2009-11-02T08:00:00Z

Pediatric Crohn's disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohn's disease (CD) is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP) have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues). The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF〈 and IFN© induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation.

Screening for Microsatellite Instability Identifies Frequent 3′-Untranslated Region Mutation of the RB1-Inducible Coiled-Coil 1 Gene in Colon Tumors

2009-11-02T08:00:00Z

Background

Coding region microsatellite instability (MSI) results in loss of gene products and promotion of microsatellite-unstable (MSI-H) carcinogenesis. Recent studies have indicated that MSI within 3′-untranslated regions (3′UTRs) may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3′UTR microsatellites (MSs) in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae.

Methodology/Principal Findings

In order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites). All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r = 0.86, p = 7.2×10⁻¹³). 3′UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%), NUAK family SNF1-like kinase 1(NUAK1, 31.0%), and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%). An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3′UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3′UTR MSI (9.0-fold; p = 3.6×10⁻⁴).

Conclusions

This mutational survey of well-characterized short 3′UTR MSs confirms that MSI incidence in MSI-H colorectal tumors correlates with MS length, but not with sequence conservation level or distance from other genetic elements. This study also identifies RB1CC1 as a novel target of frequent mutation and aberrant upregulation in MSI-H colorectal tumors. The predicted loss of a microRNA-accessible structure in mutant RB1CC1 RNA fits the hypothesis that 3′UTR MSI involves in aberrant RB1CC1 posttranscriptional upregulation. Further direct assessments are indicated to investigate this possibility.

Analysis of Gene Expression and Physiological Responses in Three Mexican Maize Landraces under Drought Stress and Recovery Irrigation

2009-10-30T07:00:00Z

Background

Drought is one of the major constraints for plant productivity worldwide. Different mechanisms of drought-tolerance have been reported for several plant species including maize. However, the differences in global gene expression between drought-tolerant and susceptible genotypes and their relationship to physiological adaptations to drought are largely unknown. The study of the differences in global gene expression between tolerant and susceptible genotypes could provide important information to design more efficient breeding programs to produce maize varieties better adapted to water limiting conditions.

Methodology/Principal Findings

Changes in physiological responses and gene expression patterns were studied under drought stress and recovery in three Mexican maize landraces which included two drought tolerant (Cajete criollo and Michoacán 21) and one susceptible (85-2) genotypes. Photosynthesis, stomatal conductance, soil and leaf water potentials were monitored throughout the experiment and microarray analysis was carried out on transcripts obtained at 10 and 17 days following application of stress and after recovery irrigation. The two tolerant genotypes show more drastic changes in global gene expression which correlate with different physiological mechanisms of adaptation to drought. Differences in the kinetics and number of up- and down-regulated genes were observed between the tolerant and susceptible maize genotypes, as well as differences between the two tolerant genotypes. Interestingly, the most dramatic differences between the tolerant and susceptible genotypes were observed during recovery irrigation, suggesting that the tolerant genotypes activate mechanisms that allow more efficient recovery after a severe drought.

Conclusions/Significance

A correlation between levels of photosynthesis and transcription under stress was observed and differences in the number, type and expression levels of transcription factor families were also identified under drought and recovery between the three maize landraces. Gene expression analysis suggests that the drought tolerant landraces have a greater capacity to rapidly modulate more genes under drought and recovery in comparison to the susceptible landrace. Modulation of a greater number of differentially expressed genes of different TF gene families is an important characteristic of the tolerant genotypes. Finally, important differences were also noted between the tolerant landraces that underlie different mechanisms of achieving tolerance.

A Wireless Lingual Feedback Device to Reduce Overpressures in Seated Posture: A Feasibility Study

2009-10-30T07:00:00Z

Background

Pressure sores are localized injuries to the skin and underlying tissues and are mainly resulting from overpressure. Paraplegic peoples are particularly subjects to pressure sores because of long-time seated postures and sensory deprivation at the lower limbs.

Methodology/Principal Findings

Here we report outcomes of a feasibility trial involving a biofeedback system aimed at reducing buttock overpressure whilst an individual is seated. The system consists of (1) pressure sensors, (2) a laptop coupling sensors and actuator (3) a wireless Tongue Display Unit (TDU) consisting of a circuit embedded in a dental retainer with electrodes put in contact with the tongue. The principle consists in (1) detecting overpressures in people who are seated over long periods of time, (2) estimating a postural change that could reduce these overpressures and (3) communicating this change through directional information transmitted by the TDU.Twenty-four healthy subjects voluntarily participated in this study. Twelve healthy subjects initially formed the experimental group (EG) and were seated on a chair with the wireless TDU inside their mouth. They were asked to follow TDU orders that were randomly spread throughout the session. They were evaluated during two experimental sessions during which 20 electro-stimulations were sent. Twelve other subjects, added retrospectively, formed the control group (CG). These subjects participated in one session of the same experiment without any biofeedback.Three dependent variables were computed: (1) the ability of subjects to reach target posture (EG versus CG), (2) high pressure reductions after a biofeedback (EG versus CG) and (3) the level of these reductions relative to their initial values (EG only). Results show (1) that EG reached target postures in 90.2% of the trials, against 5,3% in the CG, (2) a significant reduction in overpressures in the EG compared to the CG and (3), for the EG, that the higher the initial pressures were, the more they were decreased.

Conclusions/Significance

The findings suggest that, in this trial, subjects were able to use a tongue tactile feedback system to reduce buttock overpressure while seated. Further evaluation of this system on paraplegic subjects remains to be done.

Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma

2009-10-30T07:00:00Z

Background

The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients.

Principal Findings

Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 γ-carboxylated, suggesting these molecules to be active in vivo.

Significance

These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.

Structure-Function Investigation of Vsp Serotypes of the Spirochete Borrelia hermsii

2009-10-30T07:00:00Z

Background

Relapsing fever (RF) spirochetes are notable for multiphasic antigenic variation of polymorphic outer membrane lipoproteins, a phenomenon responsible for immune evasion. An additional role in tissue localization is suggested by the finding that isogenic serotypes 1 (Bt1) and 2 (Bt2) of the RF spirochete Borrelia turicatae, which differ only in the Vsp they express, exhibit marked differences in clinical disease severity and tissue localization during infection.

Methodology/Principal Findings

Here we used known vsp DNA sequences encoding for B. turicatae and Borrelia hermsii Vsp proteins with variable regions and then studied whether there are differences in disease expression and tissue localization of their corresponding serotypes during mouse infection. For sequence and structural comparisons we focused exclusively on amino acid residues predicted to project away from the spirochetes surface, referred to as the Vsp dome. Disease severity and tissue localization were studied during persistent infection with individual or mixed serotypes in SCID mice. The results showed that all Vsp domes clustered into 3 main trunks, with the domes for B. turicatae Vsp1 (BtVsp1) and BtVsp2 clustering into separate ones. B. hermsii serotypes whose Vsp domes clustered with the BtVsp1 dome were less virulent but localized to the brain more. The BtVsp2 dome was the oddball among all and Bt2 was the only serotype that caused severe arthritis.

Conclusion/Significance

These findings indicate that there is significant variability in Vsp dome structure, disease severity, and tissue localization among serotypes of B. hermsii.

Liver Is Able to Activate Naïve CD8⁺ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

2009-10-30T07:00:00Z

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8⁺ T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8⁺ T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8⁺ T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8⁺ T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8⁺ T cell response and accounts for the dysfunctional CD8⁺ T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection.

C. elegans ATAD-3 Is Essential for Mitochondrial Activity and Development

2009-10-30T07:00:00Z

Background

Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved.

Results

Here, we characterize the Caenorhabditis elegans (C. elegans) ATAD3 homologue (ATAD-3) and investigate its importance for mitochondrial function and development. We show that ATAD-3 is highly conserved among different species and RNA mediated interference against atad-3 causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality. Investigation of mitochondrial physiology revealed a disturbance in organellar structure while biogenesis and function, as indicated by complex I and citrate synthase activities, appeared to be unaltered according to the developmental stage. Nevertheless, we observed very low complex I and citrate synthase activities in L1 larvae populations in comparison to higher larval and adult stages. Our findings indicate that atad-3(RNAi) animals arrest at developmental stages with low mitochondrial activity. In addition, a reduced intestinal fat storage and low lysosomal content after depletion of ATAD-3 suggests a central role of this protein for metabolic activity.

Conclusions

In summary, our data clearly indicate that ATAD-3 is essential for C. elegans development in vivo. Moreover, our results suggest that the protein is important for the upregulation of mitochondrial activity during the transition to higher larval stages.

The Role of UPF0157 in the Folding of M. tuberculosis Dephosphocoenzyme A Kinase and the Regulation of the Latter by CTP

2009-10-30T07:00:00Z

Background

Targeting the biosynthetic pathway of Coenzyme A (CoA) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final enzymes of CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme A kinase (CoaE) as a potential drug target.

Methodology/Principal Findings

We report here a complete biochemical and biophysical characterization of the M. tuberculosis CoaE, an enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a dephosphocoenzyme A kinase with its substrates, dephosphocoenzyme A and ATP; its product, CoA and an intrinsic yet novel inhibitor, CTP, which helps modulate the enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and ATP following in tow. Kinetic and ITC experiments demonstrate that though CTP binds strongly to the enzyme, it is unable to participate in DCoA phosphorylation. We report that CTP actually inhibits the enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate kinases, deoxycytidine kinases, and cytidylate kinases as close homologs. Docking of DCoA and CTP to CoaE shows that both ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length enzyme.

Conclusions/Significance

In view of the evidence presented, it is imperative to assign a greater role to the last enzyme of Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.

Zicam-Induced Damage to Mouse and Human Nasal Tissue

2009-10-30T07:00:00Z

Intranasal medications are used to treat various nasal disorders. However, their effects on olfaction remain unknown. Zicam (zinc gluconate; Matrixx Initiatives, Inc), a homeopathic substance marketed to alleviate cold symptoms, has been implicated in olfactory dysfunction. Here, we investigated Zicam and several common intranasal agents for their effects on olfactory function. Zicam was the only substance that showed significant cytotoxicity in both mouse and human nasal tissue. Specifically, Zicam-treated mice had disrupted sensitivity of olfactory sensory neurons to odorant stimulation and were unable to detect novel odorants in behavioral testing. These findings were long-term as no recovery of function was observed after two months. Finally, human nasal explants treated with Zicam displayed significantly elevated extracellular lactate dehydrogenase levels compared to saline-treated controls, suggesting severe necrosis that was confirmed on histology. Our results demonstrate that Zicam use could irreversibly damage mouse and human nasal tissue and may lead to significant smell dysfunction.

Dynamic and Physical Clustering of Gene Expression during Epidermal Barrier Formation in Differentiating Keratinocytes

2009-10-30T07:00:00Z

The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease.

Identification, Isolation and Expansion of Myoendothelial Cells Involved in Leech Muscle Regeneration

2009-10-30T07:00:00Z

Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells.

Versatile Assays for High Throughput Screening for Activators or Inhibitors of Intracellular Proteases and Their Cellular Regulators

2009-10-30T07:00:00Z

Background

Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation.

Methods & Findings

We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.

Conclusions

Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

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