PLoS ONE Alerts: New Articles

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

2009-11-20T08:00:00Z

Background

Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information.

Results

AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins.

Conclusion

AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains.

Key Role of the GITR/GITRLigand Pathway in the Development of Murine Autoimmune Diabetes: A Potential Therapeutic Target

2009-11-20T08:00:00Z

Background

The cross-talk between pathogenic T lymphocytes and regulatory T cells (Tregs) plays a major role in the progression of autoimmune diseases. Our objective is to identify molecules and/or pathways involved in this interaction and representing potential targets for innovative therapies. Glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand are key players in the T effector/Treg interaction. GITR is expressed at low levels on resting T cells and is significantly up-regulated upon activation. Constitutive high expression of GITR is detected only on Tregs. GITR interacts with its ligand mainly expressed on antigen presenting cells and endothelial cells. It has been suggested that GITR triggering activates effector T lymphocytes while inhibiting Tregs thus contributing to the amplification of immune responses. In this study, we examined the role of GITR/GITRLigand interaction in the progression of autoimmune diabetes.

Methods and Findings

Treatment of 10-day-old non-obese diabetic (NOD) mice, which spontaneously develop diabetes, with an agonistic GITR-specific antibody induced a significant acceleration of disease onset (80% at 12 weeks of age). This activity was not due to a decline in the numbers or functional capacity of CD4⁺CD25⁺Foxp3⁺ Tregs but rather to a major activation of ‘diabetogenic’ T cells. This conclusion was supported by results showing that anti-GITR antibody exacerbates diabetes also in CD28^−/− NOD mice, which lack Tregs. In addition, treatment of NOD mice, infused with the diabetogenic CD4⁺BDC2.5 T cell clone, with GITR-specific antibody substantially increased their migration, proliferation and activation within the pancreatic islets and draining lymph nodes. As a mirror image, blockade of the GITR/GITRLigand pathway using a neutralizing GITRLigand-specific antibody significantly protected from diabetes even at late stages of disease progression. Experiments using the BDC2.5 T cell transfer model suggested that the GITRLigand antibody acted by limiting the homing and proliferation of pathogenic T cells in pancreatic lymph nodes.

Conclusion

GITR triggering plays an important costimulatory role on diabetogenic T cells contributing to the development of autoimmune responses. Therefore, blockade of the GITR/GITRLigand pathway appears as a novel promising clinically oriented strategy as GITRLigand-specific antibody applied at an advanced stage of disease progression can prevent overt diabetes.

Heterosubtype Neutralizing Responses to Influenza A (H5N1) Viruses Are Mediated by Antibodies to Virus Haemagglutinin

2009-11-20T08:00:00Z

Background

It is increasingly clear that influenza A infection induces cross-subtype neutralizing antibodies that may potentially confer protection against zoonotic infections. It is unclear whether this is mediated by antibodies to the neuraminidase (NA) or haemagglutinin (HA). We use pseudoviral particles (H5pp) coated with H5 haemagglutinin but not N1 neuraminidase to address this question. In this study, we investigate whether cross-neutralizing antibodies in persons unexposed to H5N1 is reactive to the H5 haemagglutinin.

Methodology/Principal Findings

We measured H5-neutralization antibody titers pre- and post-vaccination using the H5N1 micro-neutralization test (MN) and H5pp tests in subjects given seasonal vaccines and in selected sera from European elderly volunteers in a H5N1 vaccine trial who had detectable pre-vaccination H5N1 MN antibody titers. We found detectable (titer ≥20) H5N1 neutralizing antibodies in a minority of pre-seasonal vaccine sera and evidence of a serological response to H5N1 in others after seasonal influenza vaccination. There was excellent correlation in the antibody titers between the H5N1 MN and H5pp tests. Similar correlations were found between MN and H5pp in the pre-vaccine sera from the cohort of H5N1 vaccine trial recipients.

Conclusions/Significance

Heterosubtype neutralizing antibody to H5N1 in healthy volunteers unexposed to H5N1 is mediated by cross-reaction to the H5 haemagglutinin.

Akt and SHIP Modulate Francisella Escape from the Phagosome and Induction of the Fas-Mediated Death Pathway

2009-11-20T08:00:00Z

Francisella tularensis infects macrophages and escapes phago-lysosomal fusion to replicate within the host cytosol, resulting in host cell apoptosis. Here we show that the Fas-mediated death pathway is activated in infected cells and correlates with escape of the bacterium from the phagosome and the bacterial burden. Our studies also demonstrate that constitutive activation of Akt, or deletion of SHIP, promotes phago-lysosomal fusion and limits bacterial burden in the host cytosol, and the subsequent induction of Fas expression and cell death. Finally, we show that phagosomal escape/intracellular bacterial burden regulate activation of the transcription factors sp1/sp3, leading to Fas expression and cell death. These data identify for the first time host cell signaling pathways that regulate the phagosomal escape of Francisella, leading to the induction of Fas and subsequent host cell death.

TLR–Dependent Control of Francisella tularensis Infection and Host Inflammatory Responses

2009-11-20T08:00:00Z

Background

Francisella tularensis is the causative agent of tularemia and is classified as a Category A select agent. Recent studies have implicated TLR2 as a critical element in the host protective response to F. tularensis infection, but questions remain about whether TLR2 signaling dominates the response in all circumstances and with all species of Francisella and whether F. tularensis PAMPs are predominantly recognized by TLR2/TLR1 or TLR2/TLR6. To address these questions, we have explored the role of Toll-like receptors (TLRs) in the host response to infections with F. tularensis Live Vaccine Strain (LVS) and F. tularensis subspecies (subsp.) novicida in vivo.

Methodology/Principal Findings

C57BL/6 (B6) control mice and TLR– or MyD88-deficient mice were infected intranasally (i.n.) or intradermally (i.d.) with F. tularensis LVS or with F. tularensis subsp. novicida. B6 mice survived >21 days following infection with LVS by both routes and survival of TLR1^−/−, TLR4^−/−, and TLR6^−/− mice infected i.n. with LVS was equivalent to controls. Survival of TLR2^−/− and MyD88^−/− mice, however, was significantly reduced compared to B6 mice, regardless of the route of infection or the subspecies of F. tularensis. TLR2^−/− and MyD88^−/− mice also showed increased bacterial burdens in lungs, liver, and spleen compared to controls following i.n. infection. Primary macrophages from MyD88^−/− and TLR2^−/− mice were significantly impaired in the ability to secrete TNF and other pro-inflammatory cytokines upon ex vivo infection with LVS. TNF expression was also impaired in vivo as demonstrated by analysis of bronchoalveolar lavage fluid and by in situ immunofluorescent staining.

Conclusions/Significance

We conclude from these studies that TLR2 and MyD88, but not TLR4, play critical roles in the innate immune response to F. tularensis infection regardless of the route of infection or the subspecies. Moreover, signaling through TLR2 does not depend exclusively on TLR1 or TLR6 during F. tularensis LVS infection.

A Mechanistic Niche Model for Measuring Species' Distributional Responses to Seasonal Temperature Gradients

2009-11-20T08:00:00Z

Niche theory is central to understanding how species respond geographically to climate change. It defines a species' realized niche in a biological community, its fundamental niche as determined by physiology, and its potential niche—the fundamental niche in a given environment or geographic space. However, most predictions of the effects of climate change on species' distributions are limited to correlative models of the realized niche, which assume that species are in distributional equilibrium with respect to the variables or gradients included in the model. Here, I present a mechanistic niche model that measures species' responses to major seasonal temperature gradients that interact with the physiology of the organism. I then use lethal physiological temperatures to parameterize the model for bird species in North and South America and show that most focal bird species are not in direct physiological equilibrium with the gradients. Results also show that most focal bird species possess broad thermal tolerances encompassing novel climates that could become available with climate change. I conclude with discussion of how mechanistic niche models may be used to (i) gain insights into the processes that cause species to respond to climate change and (ii) build more accurate correlative distribution models in birds and other species.

Modulation of Outer Hair Cell Electromotility by Cochlear Supporting Cells and Gap Junctions

2009-11-20T08:00:00Z

Outer hair cell (OHC) or prestin-based electromotility is an active cochlear amplifier in the mammalian inner ear that can increase hearing sensitivity and frequency selectivity. In situ, Deiters supporting cells are well-coupled by gap junctions and constrain OHCs standing on the basilar membrane. Here, we report that both electrical and mechanical stimulations in Deiters cells (DCs) can modulate OHC electromotility. There was no direct electrical conductance between the DCs and the OHCs. However, depolarization in DCs reduced OHC electromotility associated nonlinear capacitance (NLC) and distortion products. Increase in the turgor pressure of DCs also shifted OHC NLC to the negative voltage direction. Destruction of the cytoskeleton in DCs or dissociation of the mechanical-coupling between DCs and OHCs abolished these effects, indicating the modulation through the cytoskeleton activation and DC-OHC mechanical coupling rather than via electric field potentials. We also found that changes in gap junctional coupling between DCs induced large membrane potential and current changes in the DCs and shifted OHC NLC. Uncoupling of gap junctions between DCs shifted NLC to the negative direction. These data indicate that DCs not only provide a physical scaffold to support OHCs but also can directly modulate OHC electromotility through the DC-OHC mechanical coupling. Our findings reveal a new mechanism of cochlear supporting cells and gap junctional coupling to modulate OHC electromotility and eventually hearing sensitivity in the inner ear.

A Spectrum of Severe Familial Liver Disorders Associate with Telomerase Mutations

2009-11-20T08:00:00Z

Background

Telomerase is an enzyme specialized in maintaining telomere lengths in highly proliferative cells. Loss-of-function mutations cause critical telomere shortening and are associated with the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia and with idiopathic pulmonary fibrosis. Here, we sought to determine the spectrum of clinical manifestations associated with telomerase loss-of-function mutations.

Methodology/Principal Findings

Sixty-nine individuals from five unrelated families with a variety of hematologic, hepatic, and autoimmune disorders were screened for telomerase complex gene mutations; leukocyte telomere length was measured by flow fluorescence in situ hybridization in mutation carriers and some non-carriers; the effects of the identified mutations on telomerase activity were determined; and genetic and clinical data were correlated. In six generations of a large family, a loss-of-function mutation in the telomerase enzyme gene TERT associated with severe telomere shortening and a range of hematologic manifestations, from macrocytosis to acute myeloid leukemia, with severe liver diseases marked by fibrosis and inflammation, and one case of idiopathic pulmonary fibrosis but not with autoimmune disorders. Additionally, we identified four unrelated families in which loss-of-function TERC or TERT gene mutations tracked with marrow failure, pulmonary fibrosis, and a spectrum of liver disorders.

Conclusions/Significance

These results indicate that heterozygous telomerase loss-of-function mutations associate with but are not determinant of a large spectrum of hematologic and liver abnormalities, with the latter sometimes occurring in the absence of marrow failure. Our findings, along with the link between pulmonary fibrosis and telomerase mutations, also suggest a common pathogenic mechanism for fibrotic diseases in which defective telomere repair plays important role.

Genome-Wide Scan for Signatures of Human Population Differentiation and Their Relationship with Natural Selection, Functional Pathways and Diseases

2009-11-20T08:00:00Z

Genetic differences both between individuals and populations are studied for their evolutionary relevance and for their potential medical applications. Most of the genetic differentiation among populations are caused by random drift that should affect all loci across the genome in a similar manner. When a locus shows extraordinary high or low levels of population differentiation, this may be interpreted as evidence for natural selection. The most used measure of population differentiation was devised by Wright and is known as fixation index, or F_ST. We performed a genome-wide estimation of F_ST on about 4 millions of SNPs from HapMap project data. We demonstrated a heterogeneous distribution of F_ST values between autosomes and heterochromosomes. When we compared the F_ST values obtained in this study with another evolutionary measure obtained by comparative interspecific approach, we found that genes under positive selection appeared to show low levels of population differentiation. We applied a gene set approach, widely used for microarray data analysis, to detect functional pathways under selection. We found that one pathway related to antigen processing and presentation showed low levels of F_ST, while several pathways related to cell signalling, growth and morphogenesis showed high F_ST values. Finally, we detected a signature of selection within genes associated with human complex diseases. These results can help to identify which process occurred during human evolution and adaptation to different environments. They also support the hypothesis that common diseases could have a genetic background shaped by human evolution.

IL-6 Deficiency Attenuates Murine Diet-Induced Non-Alcoholic Steatohepatitis

2009-11-20T08:00:00Z

Background

The role of inflammation in the pathogenesis of non-alcoholic steatohepatitis (NASH), a common cause of liver disease, is still poorly understood. This study aimed at assessing the involvement of a major inflammatory cytokine, IL-6, in NASH.

Materials and Methods

Steatohepatitis was induced by feeding wild-type or IL-6^−/− mice for 5 weeks with a methionine and choline-deficient (MCD) diet.

Results

Whereas MCD diet-induced weight loss and decreases in serum glucose, cholesterol and triglyceride levels were similar in both genotypes, serum alanine aminotransferase was less elevated in IL-6^−/− mice than in wild-type animals. Despite having a comparable liver steatosis score, IL-6-deficient mice exhibited less lobular inflammation than their wild-type littermates. Liver gene expression of TGF-β and MCP-1 was also strongly attenuated in mutant mice; a more modest reduction was observed for PPAR-γ and F4/80 transcripts as well as proteins. Chromatographic analysis of liver lipids demonstrated that MCD diet induced in normal and mutant mice a similar decrease in the ratio of phosphatidylcholine to phosphatidylethanolamine. However, the diet-induced increase in the levels of sphingomyelin and ceramide was less important in IL-6^−/− mice.

Conclusion

Altogether, these results indicate that IL-6 deficiency does not block the development of NASH; yet, IL-6 plays a critical role in the accompanying liver inflammation.

Lrp4, a Novel Receptor for Dickkopf 1 and Sclerostin, Is Expressed by Osteoblasts and Regulates Bone Growth and Turnover In Vivo

2009-11-20T08:00:00Z

Lrp4 is a multifunctional member of the low density lipoprotein-receptor gene family and a modulator of extracellular cell signaling pathways in development. For example, Lrp4 binds Wise, a secreted Wnt modulator and BMP antagonist. Lrp4 shares structural elements within the extracellular ligand binding domain with Lrp5 and Lrp6, two established Wnt co-receptors with important roles in osteogenesis. Sclerostin is a potent osteocyte secreted inhibitor of bone formation that directly binds Lrp5 and Lrp6 and modulates both BMP and Wnt signaling. The anti-osteogenic effect of sclerostin is thought to be mediated mainly by inhibition of Wnt signaling through Lrp5/6 within osteoblasts. Dickkopf1 (Dkk1) is another potent soluble Wnt inhibitor that binds to Lrp5 and Lrp6, can displace Lrp5-bound sclerostin and is itself regulated by BMPs. In a recent genome-wide association study of bone mineral density a significant modifier locus was detected near the SOST gene at 17q21, which encodes sclerostin. In addition, nonsynonymous SNPs in the LRP4 gene were suggestively associated with bone mineral density. Here we show that Lrp4 is expressed in bone and cultured osteoblasts and binds Dkk1 and sclerostin in vitro. MicroCT analysis of Lrp4 deficient mutant mice revealed shortened total femur length, reduced cortical femoral perimeter, and reduced total femur bone mineral content (BMC) and bone mineral density (BMD). Lumbar spine trabecular bone volume per total volume (BV/TV) was significantly reduced in the mutants and the serum and urinary bone turnover markers alkaline phosphatase, osteocalcin and desoxypyridinoline were increased. We conclude that Lrp4 is a novel osteoblast expressed Dkk1 and sclerostin receptor with a physiological role in the regulation of bone growth and turnover, which is likely mediated through its function as an integrator of Wnt and BMP signaling pathways.

Early-Onset and Robust Amyloid Pathology in a New Homozygous Mouse Model of Alzheimer's Disease

2009-11-20T08:00:00Z

Background

Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral β-amyloidosis, one of the characteristic hallmarks of Alzheimer's disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues.

Methodology/Principal Findings

The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APP_swe) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral β-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a γ-secretase inhibitor we show a dose dependent reduction of soluble amyloid β levels in the brain.

Conclusions

ARTE10 mice develop a cerebral β-amyloidosis closely resembling the β-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD.

CRX Is a Diagnostic Marker of Retinal and Pineal Lineage Tumors

2009-11-20T08:00:00Z

Background

CRX is a homeobox transcription factor whose expression and function is critical to maintain retinal and pineal lineage cells and their progenitors. To determine the biologic and diagnostic potential of CRX in human tumors of the retina and pineal, we examined its expression in multiple settings.

Methodology/Principal Findings

Using situ hybridization and immunohistochemistry we show that Crx RNA and protein expression are exquisitely lineage restricted to retinal and pineal cells during normal mouse and human development. Gene expression profiling analysis of a wide range of human cancers and cancer cell lines also supports that CRX RNA is highly lineage restricted in cancer. Immunohistochemical analysis of 22 retinoblastomas and 13 pineal parenchymal tumors demonstrated strong expression of CRX in over 95% of these tumors. Importantly, CRX was not detected in the majority of tumors considered in the differential diagnosis of pineal region tumors (n = 78). The notable exception was medulloblastoma, 40% of which exhibited CRX expression in a heterogeneous pattern readily distinguished from that seen in retino-pineal tumors.

Conclusions/Significance

These findings describe new potential roles for CRX in human cancers and highlight the general utility of lineage restricted transcription factors in cancer biology. They also identify CRX as a sensitive and specific clinical marker and a potential lineage dependent therapeutic target in retinoblastoma and pineoblastoma.

Motor and Linguistic Linking of Space and Time in the Cerebellum

2009-11-20T08:00:00Z

Background

Recent literature documented the presence of spatial-temporal interactions in the human brain. The aim of the present study was to verify whether representation of past and future is also mapped onto spatial representations and whether the cerebellum may be a neural substrate for linking space and time in the linguistic domain. We asked whether processing of the tense of a verb is influenced by the space where response takes place and by the semantics of the verb.

Principal Findings

Responses to past tense were facilitated in the left space while responses to future tense were facilitated in the right space. Repetitive transcranial magnetic stimulation (rTMS) of the right cerebellum selectively slowed down responses to future tense of action verbs; rTMS of both cerebellar hemispheres decreased accuracy of responses to past tense in the left space and to future tense in the right space for non-verbs, and to future tense in the right space for state verbs.

Conclusions

The results suggest that representation of past and future is mapped onto spatial formats and that motor action could represent the link between spatial and temporal dimensions. Right cerebellar, left motor brain networks could be part of the prospective brain, whose primary function is to use past experiences to anticipate future events. Both cerebellar hemispheres could play a role in establishing the grammatical rules for verb conjugation.

p38γ Mitogen-Activated Protein Kinase Is a Key Regulator in Skeletal Muscle Metabolic Adaptation in Mice

2009-11-20T08:00:00Z

Regular endurance exercise induces skeletal muscle contractile and metabolic adaptations, conferring salutary health benefits, such as protection against the metabolic syndrome. The plasticity of skeletal muscle has been extensively investigated, but how the adaptive processes are precisely controlled is largely unknown. Using muscle-specific gene deletion in mice, we now show that p38γ mitogen-activated protein kinase (MAPK), but not p38α and p38β, is required for endurance exercise-induced mitochondrial biogenesis and angiogenesis, whereas none of the p38 isoforms are required for IIb-to-IIa fiber-type transformation. These phenotypic findings were further supported by microarray and real-time PCR analyses revealing contractile activity-dependent p38γ target genes, including peroxisome proliferator-activated receptor γ co-activator-1α (Pgc-1α) and vascular endothelial growth factor (Vegf), in skeletal muscle following motor nerve stimulation. Gene transfer-mediated overexpression of a dominant negative form of p38γ, but not that of p38α or p38β, blocked motor nerve stimulation-induced Pgc-1α transcription. These findings provide direct evidence for an obligated role of p38γ MAPK-PGC-1α regulatory axis in endurance exercise-induced metabolic adaptation, but not contractile adaptation, in skeletal muscle.

Divergent Mechanisms Controlling Hypoxic Sensitivity and Lifespan by the DAF-2/Insulin/IGF-Receptor Pathway

2009-11-20T08:00:00Z

Organisms and their cells vary greatly in their tolerance of low oxygen environments (hypoxia). A delineation of the determinants of hypoxia tolerance is incomplete, despite intense interest for its implications in diseases such as stroke and myocardial infarction. The insulin/IGF-1 receptor (IGFR) signaling pathway controls survival of Caenorhabditis elegans from a variety of stressors including aging, hyperthermia, and hypoxia. daf-2 encodes a C. elegans IGFR homolog whose primary signaling pathway modulates the activity of the FOXO transcription factor DAF-16. DAF-16 regulates the transcription of a large number of genes, some of which have been shown to control aging. To identify genes that selectively regulate hypoxic sensitivity, we compared the whole-organismal transcriptomes of three daf-2 reduction-of-function alleles, all of which are hypoxia resistant, thermotolerant, and long lived, but differ in their rank of severities for these phenotypes. The transcript levels of 172 genes were increased in the most hypoxia resistant daf-2 allele, e1370, relative to the other alleles whereas transcripts from only 10 genes were decreased in abundance. RNAi knockdown of 6 of the 10 genes produced a significant increase in organismal survival after hypoxic exposure as would be expected if down regulation of these genes by the e1370 mutation was responsible for hypoxia resistance. However, RNAi knockdown of these genes did not prolong lifespan. These genes definitively separate the mechanisms of hypoxic sensitivity and lifespan and identify biological strategies to survive hypoxic injury.

Combination of Real-Value Smell and Metaphor Expression Aids Yeast Detection

2009-11-20T08:00:00Z

Background

Smell provides important information about the quality of food and drink. Most well-known for their expertise in wine tasting, sommeliers sniff out the aroma of wine and describe them using beautiful metaphors. In contrast, electronic noses, devices that mimic our olfactory recognition system, also detect smells using their sensors but describe them using electronic signals. These devices have been used to judge the freshness of food or detect the presence of pathogenic microorganisms. However, unlike information from gas chromatography, it is difficult to compare odour information collected by these devices because they are made for smelling specific smells and their data are relative intensities.

Methodology

Here, we demonstrate the use of an absolute-value description method using known smell metaphors, and early detection of yeast using the method.

Conclusions

This technique may help distinguishing microbial-contamination of food products earlier, or improvement of the food-product qualities.

Stem Loop Sequences Specific to Transposable Element IS605 Are Found Linked to Lipoprotein Genes in Borrelia Plasmids

2009-11-20T08:00:00Z

Background

Plasmids of Borrelia species are dynamic structures that contain a large number of repetitive genes, gene fragments, and gene fusions. In addition, the transposable element IS605/200 family, as well as degenerate forms of this IS element, are prevalent. In Helicobacter pylori, flanking regions of the IS605 transposase gene contain sequences that fold into identical small stem loops. These function in transposition at the single-stranded DNA level.

Methodology/Principal Findings

In work reported here, bioinformatics techniques were used to scan Borrelia plasmid genomes for IS605 transposable element specific stem loop sequences. Two variant stem loop motifs are found in the left and right flanking regions of the transposase gene. Both motifs appear to have dispersed in plasmid genomes and are found “free-standing” and phylogenetically conserved without the associated IS605 transposase gene or the adjacent flanking sequence. Importantly, IS605 specific stem loop sequences are also found at the 3′ ends of lipoprotein genes (PFam12 and PFam60), however the left and right sequences appear to develop their own evolutionary patterns. The lipoprotein gene-linked left stem loop sequences maintain the IS605 stem loop motif in orthologs but only at the RNA level. These show mutations whereby variants fold into phylogenetically conserved RNA-type stem loops that contain the wobble non-Watson-Crick G-U base-pairing. The right flanking sequence is associated with the family lipoprotein-1 genes. A comparison of homologs shows that the IS605 stem loop motif rapidly dissipates, but a more elaborate secondary structure appears to develop in its place.

Conclusions/Significance

Stem loop sequences specific to the transposable element IS605 are present in plasmid regions devoid of a transposase gene and significantly, are found linked to lipoprotein genes in Borrelia plasmids. These sequences are evolutionarily conserved and/or structurally developed in an RNA format. The findings show that IS605 stem loop sequences are multifaceted and are selectively conserved during evolution when the transposable element dissipates.

Absence of ERRα in Female Mice Confers Resistance to Bone Loss Induced by Age or Estrogen-Deficiency

2009-11-20T08:00:00Z

Background

ERRα is an orphan member of the nuclear hormone receptor superfamily, which acts as a transcription factor and is involved in various metabolic processes. ERRα is also highly expressed in ossification zones during mouse development as well as in human bones and cell lines. Previous data have shown that this receptor up-modulates the expression of osteopontin, which acts as an inhibitor of bone mineralization and whose absence results in resistance to ovariectomy-induced bone loss. Altogether this suggests that ERRα may negatively regulate bone mass and could impact on bone fragility that occurs in the absence of estrogens.

Methods/Principal Findings

In this report, we have determined the in vivo effect of ERRα on bone, using knock-out mice. Relative to wild type animals, female ERRαKO bones do not age and are resistant to bone loss induced by estrogen-withdrawal. Strikingly male ERRαKO mice are indistinguishable from their wild type counterparts, both at the unchallenged or gonadectomized state. Using primary cell cultures originating from ERRαKO bone marrow, we also show that ERRα acts as an inhibitor of osteoblast differentiation.

Conclusion/Significance

Down-regulating ERRα could thus be beneficial against osteoporosis.

On Theoretical Models of Gene Expression Evolution with Random Genetic Drift and Natural Selection

2009-11-20T08:00:00Z

Background

The relative contributions of natural selection and random genetic drift are a major source of debate in the study of gene expression evolution, which is hypothesized to serve as a bridge from molecular to phenotypic evolution. It has been suggested that the conflict between views is caused by the lack of a definite model of the neutral hypothesis, which can describe the long-run behavior of evolutionary change in mRNA abundance. Therefore previous studies have used inadequate analogies with the neutral prediction of other phenomena, such as amino acid or nucleotide sequence evolution, as the null hypothesis of their statistical inference.

Methodology/Principal Findings

In this study, we introduced two novel theoretical models, one based on neutral drift and the other assuming natural selection, by focusing on a common property of the distribution of mRNA abundance among a variety of eukaryotic cells, which reflects the result of long-term evolution. Our results demonstrated that (1) our models can reproduce two independently found phenomena simultaneously: the time development of gene expression divergence and Zipf's law of the transcriptome; (2) cytological constraints can be explicitly formulated to describe long-term evolution; (3) the model assuming that natural selection optimized relative mRNA abundance was more consistent with previously published observations than the model of optimized absolute mRNA abundances.

Conclusions/Significance

The models introduced in this study give a formulation of evolutionary change in the mRNA abundance of each gene as a stochastic process, on the basis of previously published observations. This model provides a foundation for interpreting observed data in studies of gene expression evolution, including identifying an adequate time scale for discriminating the effect of natural selection from that of random genetic drift of selectively neutral variations.

Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

2009-11-20T08:00:00Z

Background

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.

Methodology/Principal Findings

In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space.

Conclusions/Significance

These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.

Gene Expression Profiling during Early Acute Febrile Stage of Dengue Infection Can Predict the Disease Outcome

2009-11-19T08:00:00Z

Background

We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples.

Methodology/Principal Findings

The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity: classic dengue fever or dengue hemorrhagic fever (DHF) samples were compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2–3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels.

Conclusions/Significance

Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection.

Synergy between Repellents and Organophosphates on Bed Nets: Efficacy and Behavioural Response of Natural Free-Flying An. gambiae Mosquitoes

2009-11-19T08:00:00Z

Background

Chemicals are used on bed nets in order to prevent infected bites and to kill aggressive malaria vectors. Because pyrethroid resistance has become widespread in the main malaria vectors, research for alternative active ingredients becomes urgent. Mixing a repellent and a non-pyrethroid insecticide seemed to be a promising tool as mixtures in the laboratory showed the same features as pyrethroids.

Methodology/Principal Findings

We present here the results of two trials run against free-flying Anopheles gambiae populations comparing the effects of two insect repellents (either DEET or KBR 3023, also known as icaridin) and an organophosphate insecticide at low-doses (pirimiphos-methyl, PM) used alone and in combination on bed nets. We showed that mixtures of PM and the repellents induced higher exophily, blood feeding inhibition and mortality among wild susceptible and resistant malaria vectors than compounds used alone. Nevertheless the synergistic interactions are only involved in the high mortality induced by the two mixtures.

Conclusion

These field trials argue in favour of the strategy of mixing repellent and organophosphate on bed nets to better control resistant malaria vectors.

Catastrophic NAD⁺ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

2009-11-19T08:00:00Z

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD⁺ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD⁺ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD⁺-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD⁺ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.

Socioeconomic Predictors of Cognition in Ugandan Children: Implications for Community Interventions

2009-11-19T08:00:00Z

Background

Several interventions to improve cognition in at risk children have been suggested. Identification of key variables predicting cognition is necessary to guide these interventions. This study was conducted to identify these variables in Ugandan children and guide such interventions.

Methods

A cohort of 89 healthy children (45 females) aged 5 to 12 years old were followed over 24 months and had cognitive tests measuring visual spatial processing, memory, attention and spatial learning administered at baseline, 6 months and 24 months. Nutritional status, child's educational level, maternal education, socioeconomic status and quality of the home environment were also measured at baseline. A multivariate, longitudinal model was then used to identify predictors of cognition over the 24 months.

Results

A higher child's education level was associated with better memory (p = 0.03), attention (p = 0.005) and spatial learning scores over the 24 months (p = 0.05); higher nutrition scores predicted better visual spatial processing (p = 0.002) and spatial learning scores (p = 0.008); and a higher home environment score predicted a better memory score (p = 0.03).

Conclusion

Cognition in Ugandan children is predicted by child's education, nutritional status and the home environment. Community interventions to improve cognition may be effective if they target multiple socioeconomic variables.

Feasibility of Provider-Initiated HIV Testing and Counselling of Tuberculosis Patients Under the TB Control Programme in Two Districts of South India

2009-11-19T08:00:00Z

Background

Provider-initiated HIV testing and counselling (PITC) is internationally recommended for tuberculosis (TB) patients, but the feasibility, effectiveness, and impact of this policy on the TB programme in India are unknown. We evaluated PITC of TB patients across two districts in India considered to have generalized HIV epidemics, Tiruchirappalli (population 2.5 million) and Mysore (population 2.8 million).

Methodology/Principal Findings

Starting June 2007, healthcare providers in both districts were instructed to ascertain HIV status for all TB patients, and refer those with unknown HIV status to the nearest Integrated Counselling and Testing Centre (ICTC)—often in the same facility—for counselling and voluntary HIV testing. All TB patients registered from June 2007 to March 2008 were followed prospectively. Field investigators assessed PITC practices and abstracted data from routine TB programme records and HIV counselling registers to determine the proportion of TB patients appropriately evaluated for HIV infection. Patient records were traced to determine the efficiency of referral links to HIV care and antiretroviral treatment (ART). Between July 2007 and March 2008, 5299 TB patients were registered in both study districts. Of the 4701 with unknown HIV status at the time of TB treatment initiation, 3368 (72%) were referred to an ICTC, and 3111 (66%) were newly tested for HIV. PITC implementation resulted in the ascertainment of HIV status for 3709/5299 (70%) of TB patients, and detected 200 cases with previously undiagnosed HIV infection. Overall, 468 (8.8%) of all registered TB patients were HIV-infected; 177 (37%) were documented to have also received any ART.

Conclusions

With implementation of PITC in India, HIV status was successfully ascertained for 70% of TB patients. Previously undiagnosed HIV-infection was detected in 6.4% of those TB patients newly tested, enabling referral for life-saving anti-retroviral treatment. ART uptake, however, was poor, suggesting that PITC implementation should include measures to strengthen and support ART referral, evaluation, and initiation.

Inhibition of PI3K-Akt Signaling Blocks Exercise-Mediated Enhancement of Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

2009-11-19T08:00:00Z

Background

Physical exercise has been shown to increase adult neurogenesis in the dentate gyrus and enhances synaptic plasticity. The antiapoptotic kinase, Akt has also been shown to be phosphorylated following voluntary exercise; however, it remains unknown whether the PI3K-Akt signaling pathway is involved in exercise-induced neurogenesis and the associated facilitation of synaptic plasticity in the dentate gyrus.

Methodology/Principal Findings

To gain insight into the potential role of this signaling pathway in exercise-induced neurogenesis and LTP in the dentate gyrus rats were infused with the PI3K inhibitor, LY294002 or vehicle control solution (icv) via osmotic minipumps and exercised in a running wheel for 10 days. Newborn cells in the dentate gyrus were date-labelled with BrdU on the last 3 days of exercise. Then, they were either returned to the home cage for 2 weeks to assess exercise-induced LTP and neurogenesis in the dentate gyrus, or were killed on the last day of exercise to assess proliferation and activation of the PI3K-Akt cascade using western blotting.

Conclusions/Significance

Exercise increases cell proliferation and promotes survival of adult-born neurons in the dentate gyrus. Immediately after exercise, we found that Akt and three downstream targets, BAD, GSK3β and FOXO1 were activated. LY294002 blocked exercise-induced phosphorylation of Akt and downstream target proteins. This had no effect on exercise-induced cell proliferation, but it abolished most of the beneficial effect of exercise on the survival of newly generated dentate gyrus neurons and prevented exercise-induced increase in dentate gyrus LTP. These results suggest that activation of the PI3 kinase-Akt signaling pathway plays a significant role via an antiapoptotic function in promoting survival of newly formed granule cells generated during exercise and the associated increase in synaptic plasticity in the dentate gyrus.

The Astronomical Orientation of Ancient Greek Temples

2009-11-19T08:00:00Z

Despite its appearing to be a simple question to answer, there has been no consensus as to whether or not the alignments of ancient Greek temples reflect astronomical intentions. Here I present the results of a survey of archaic and classical Greek temples in Sicily and compare them with temples in Greece. Using a binomial test I show strong evidence that there is a preference for solar orientations. I then speculate that differences in alignment patterns between Sicily and Greece reflect differing pressures in the expression of ethnic identity.

Epigenetic Features of Human Mesenchymal Stem Cells Determine Their Permissiveness for Induction of Relevant Transcriptional Changes by SYT-SSX1

2009-11-19T08:00:00Z

Background

A characteristic SYT–SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism.

Methodology/Principal Findings

We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression.

Conclusions/Significance

Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Metabolite Profiling Identifies Candidate Markers Reflecting the Clinical Adaptations Associated with Roux-en-Y Gastric Bypass Surgery

2009-11-19T08:00:00Z

Background

Roux-en-Y gastric bypass (RYGB) surgery is associated with weight loss, improved insulin sensitivity and glucose homeostasis, and a reduction in co-morbidities such as diabetes and coronary heart disease. To generate further insight into the numerous metabolic adaptations associated with RYGB surgery, we profiled serum metabolites before and after gastric bypass surgery and integrated metabolite changes with clinical data.

Methodology and Principal Findings

Serum metabolites were detected by gas and liquid chromatography-coupled mass spectrometry before, and 3 and 6 months after RYGB in morbidly obese female subjects (n = 14; BMI = 46.2±1.7). Subjects showed decreases in weight-related parameters and improvements in insulin sensitivity post surgery. The abundance of 48% (83 of 172) of the measured metabolites changed significantly within the first 3 months post RYGB (p<0.05), including sphingosines, unsaturated fatty acids, and branched chain amino acids. Dividing subjects into obese (n = 9) and obese/diabetic (n = 5) groups identified 8 metabolites that differed consistently at all time points and whose serum levels changed following RYGB: asparagine, lysophosphatidylcholine (C18:2), nervonic (C24:1) acid, p-Cresol sulfate, lactate, lycopene, glucose, and mannose. Changes in the aforementioned metabolites were integrated with clinical data for body mass index (BMI) and estimates for insulin resistance (HOMA-IR). Of these, nervonic acid was significantly and negatively correlated with HOMA-IR (p = 0.001, R = −0.55).

Conclusions

Global metabolite profiling in morbidly obese subjects after RYGB has provided new information regarding the considerable metabolic alterations associated with this surgical procedure. Integrating clinical measurements with metabolomics data is capable of identifying markers that reflect the metabolic adaptations following RYGB.

Genome-Wide Analysis of Immune Activation in Human T and B Cells Reveals Distinct Classes of Alternatively Spliced Genes

2009-11-19T08:00:00Z

Alternative splicing of pre-mRNA is a mechanism that increases the protein diversity of a single gene by differential exon inclusion/exclusion during post-transcriptional processing. While alternative splicing is established to occur during lymphocyte activation, little is known about the role it plays during the immune response. Our study is among the first reports of a systematic genome-wide analysis of activated human T and B lymphocytes using whole exon DNA microarrays integrating alternative splicing and differential gene expression. Purified human CD2⁺ T or CD19⁺ B cells were activated using protocols to model the early events in post-transplant allograft immunity and sampled as a function of time during the process of immune activation. Here we show that 3 distinct classes of alternatively spliced and/or differentially expressed genes change in an ordered manner as a function of immune activation. We mapped our results to function-based canonical pathways and demonstrated that some are populated by only one class of genes, like integrin signaling, while other pathways, such as purine metabolism and T cell receptor signaling, are populated by all three classes of genes. Our studies augment the current view of T and B cell activation in immunity that has been based exclusively upon differential gene expression by providing evidence for a large number of molecular networks populated as a function of time and activation by alternatively spliced genes, many of which are constitutively expressed.

Performance of an Influenza Rapid Test in Children in a Primary Healthcare Setting in Nicaragua

2009-11-19T08:00:00Z

Background

Influenza is major public health threat worldwide, yet the diagnostic accuracy of rapid tests in developing country settings is not well described.

Methodology/Principal Findings

To investigate the diagnostic accuracy of the QuickVue Influenza A+B test in a primary care setting in a developing country, we performed a prospective study of diagnostic accuracy of the QuickVue Influenza A+B test in comparison to reverse transcriptase-polymerase chain reaction (RT-PCR) in a primary healthcare setting in children aged 2 to 12 years in Managua, Nicaragua. The sensitivity and specificity of the QuickVue test compared to RT-PCR were 68.5% (95% CI 63.4, 73.3) and 98.1% (95% CI 96.9, 98.9), respectively, for children with a fever or history of a fever and cough and/or sore throat. Test performance was found to be lower on the first day that symptoms developed in comparison to test performance on days two or three of illness.

Conclusions/Significance

Our study found that the QuickVue Influenza A+B test performed as well in a developing country primary healthcare facility setting as in developed country settings.

H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

2009-11-19T08:00:00Z

Background

Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Methodology/Principal Findings

Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.

Conclusions/Significance

The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.

Phenotypic Plasticity of Mouse Spermatogonial Stem Cells

2009-11-19T08:00:00Z

Background

Spermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro.

Methodology and Principal Findings

In this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit⁻ and Kit⁺ cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo.

Conclusions/Significance

These results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.

Generation and Characterization of Fmr1 Knockout Zebrafish

2009-11-19T08:00:00Z

Fragile X syndrome (FXS) is one of the most common known causes of inherited mental retardation. The gene mutated in FXS is named FMR1, and is well conserved from human to Drosophila. In order to generate a genetic tool to study FMR1 function during vertebrate development, we generated two mutant alleles of the fmr1 gene in zebrafish. Both alleles produce no detectable Fmr protein, and produce viable and fertile progeny with lack of obvious phenotypic features. This is in sharp contrast to published results based on morpholino mediated knock-down of fmr1, reporting defects in craniofacial development and neuronal branching in embryos. These phenotypes we specifically addressed in our knock-out animals, revealing no significant deviations from wild-type animals, suggesting that the published morpholino based fmr1 phenotypes are potential experimental artifacts. Therefore, their relation to fmr1 biology is questionable and morpholino induced fmr1 phenotypes should be avoided in screens for potential drugs suitable for the treatment of FXS. Importantly, a true genetic zebrafish model is now available which can be used to study FXS and to derive potential drugs for FXS treatment.

Evidence for Anomalous Network Connectivity during Working Memory Encoding in Schizophrenia: An ICA Based Analysis

2009-11-19T08:00:00Z

Background

Numerous neuroimaging studies report abnormal regional brain activity during working memory performance in schizophrenia, but few have examined brain network integration as determined by “functional connectivity” analyses.

Methodology/Principal Findings

We used independent component analysis (ICA) to identify and characterize dysfunctional spatiotemporal networks in schizophrenia engaged during the different stages (encoding and recognition) of a Sternberg working memory fMRI paradigm. 37 chronic schizophrenia and 54 healthy age/gender-matched participants performed a modified Sternberg Item Recognition fMRI task. Time series images preprocessed with SPM2 were analyzed using ICA. Schizophrenia patients showed relatively less engagement of several distinct “normal” encoding-related working memory networks compared to controls. These encoding networks comprised 1) left posterior parietal-left dorsal/ventrolateral prefrontal cortex, cingulate, basal ganglia, 2) right posterior parietal, right dorsolateral prefrontal cortex and 3) default mode network. In addition, the left fronto-parietal network demonstrated a load-dependent functional response during encoding. Network engagement that differed between groups during recognition comprised the posterior cingulate, cuneus and hippocampus/parahippocampus. As expected, working memory task accuracy differed between groups (p<0.0001) and was associated with degree of network engagement. Functional connectivity within all three encoding-associated functional networks correlated significantly with task accuracy, which further underscores the relevance of abnormal network integration to well-described schizophrenia working memory impairment. No network was significantly associated with task accuracy during the recognition phase.

Conclusions/Significance

This study extends the results of numerous previous schizophrenia studies that identified isolated dysfunctional brain regions by providing evidence of disrupted schizophrenia functional connectivity using ICA within widely-distributed neural networks engaged for working memory cognition.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

2009-11-19T08:00:00Z

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4⁺IL-17⁺, CD4⁺IFN-γ⁺ and CD4⁺Foxp3⁺ cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4⁺IFN-γ⁺ and CD4⁺IL-17⁺ T cells, corroborating with higher levels of IFN-γ. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription

2009-11-19T08:00:00Z

Background

Over 15 inherited diseases are caused by expansion of triplet-repeats. Friedreich ataxia (FRDA) patients are homozygous for an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene. The expanded GAA triplet-repeat results in deficiency of FXN gene transcription, which is reversed via administration of histone deacetylase inhibitors indicating that transcriptional silencing is at least partially due to an epigenetic abnormality.

Methodology/Principal Findings

We found a severe depletion of the chromatin insulator protein CTCF (CCCTC-binding factor) in the 5′UTR of the FXN gene in FRDA, and coincident heterochromatin formation involving the +1 nucleosome via enrichment of H3K9me3 and recruitment of heterochromatin protein 1. We identified FAST-1 (FXN Antisense Transcript – 1), a novel antisense transcript that overlaps the CTCF binding site in the 5′UTR, which was expressed at higher levels in FRDA. The reciprocal relationship of deficient FXN transcript and higher levels of FAST-1 seen in FRDA was reproduced in normal cells via knockdown of CTCF.

Conclusions/Significance

CTCF depletion constitutes an epigenetic switch that results in increased antisense transcription, heterochromatin formation and transcriptional deficiency in FRDA. These findings provide a mechanistic basis for the transcriptional silencing of the FXN gene in FRDA, and broaden our understanding of disease pathogenesis in triplet-repeat diseases.

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

2009-11-19T08:00:00Z

Background

The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.

Methodology/Principal Findings

We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.

Conclusions/Significance

Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.

Experimental Diabetes Mellitus Exacerbates Tau Pathology in a Transgenic Mouse Model of Alzheimer's Disease

2009-11-19T08:00:00Z

Diabetes mellitus (DM) is characterized by hyperglycemia caused by a lack of insulin, insulin resistance, or both. There is increasing evidence that insulin also plays a role in Alzheimer's disease (AD) as it is involved in the metabolism of β-amyloid (Aβ) and tau, two proteins that form Aβ plaques and neurofibrillary tangles (NFTs), respectively, the hallmark lesions in AD. Here, we examined the effects of experimental DM on a pre-existing tau pathology in the pR5 transgenic mouse strain that is characterized by NFTs. pR5 mice express P301L mutant human tau that is associated with dementia. Experimental DM was induced by administration of streptozotocin (STZ), which causes insulin deficiency. We determined phosphorylation of tau, using immunohistochemistry and Western blotting. Solubility of tau was determined upon extraction with sarkosyl and formic acid, and Gallyas silver staining was employed to reveal NFTs. Insulin depletion by STZ administration in six months-old non-transgenic mice causes increased tau phosphorylation, without its deposition or NFT formation. In contrast, in pR5 mice this results in massive deposition of hyperphosphorylated, insoluble tau. Furthermore, they develop a pronounced tau-histopathology, including NFTs at this early age, while the pathology in sham-treated pR5 mice is moderate. Whereas experimental DM did not result in deposition of hyperphosphorylated tau in non-transgenic mice, a predisposition to develop a tau pathology in young pR5 mice was both sufficient and necessary to exacerbate tau deposition and NFT formation. Hence, DM can accelerate onset and increase severity of disease in individuals with a predisposition to developing tau pathology.

The Background of Mitochondrial DNA Haplogroup J Increases the Sensitivity of Leber's Hereditary Optic Neuropathy Cells to 2,5-Hexanedione Toxicity

2009-11-19T08:00:00Z

Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA) point mutations in complex I subunit genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. Recently it was also documented that the professional exposure to n-hexane might act as an exogenous trigger for LHON. Therefore, we here investigate the effect of the n-hexane neurotoxic metabolite 2,5-hexanedione (2,5-HD) on cell viability and mitochondrial function of different cell models (cybrids and fibroblasts) carrying the LHON mutations on different mtDNA haplogroups. The viability of control and LHON cybrids and fibroblasts, whose mtDNAs were completely sequenced, was assessed using the MTT assay. Mitochondrial ATP synthesis rate driven by complex I substrates was determined with the luciferine/luciferase method. Incubation with 2,5-HD caused the maximal loss of viability in control and LHON cells. The toxic effect of this compound was similar in control cells irrespective of the mtDNA background. On the contrary, sensitivity to 2,5-HD induced cell death was greatly increased in LHON cells carrying the 11778/ND4 or the 14484/ND6 mutation on haplogroup J, whereas the 11778/ND4 mutation in association with haplogroups U and H significantly improved cell survival. The 11778/ND4 mutation on haplogroup U was also more resistant to inhibition of complex I dependent ATP synthesis by 2,5-HD. In conclusion, this study shows that mtDNA haplogroups modulate the response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has broad implications for other neurodegenerative disorders such as Parkinson's disease.

Emotional Modulation of Attention: Fear Increases but Disgust Reduces the Attentional Blink

2009-11-19T08:00:00Z

Background

It is well known that facial expressions represent important social cues. In humans expressing facial emotion, fear may be configured to maximize sensory exposure (e.g., increases visual input) whereas disgust can reduce sensory exposure (e.g., decreases visual input). To investigate whether such effects also extend to the attentional system, we used the “attentional blink” (AB) paradigm. Many studies have documented that the second target (T2) of a pair is typically missed when presented within a time window of about 200–500 ms from the first to-be-detected target (T1; i.e., the AB effect). It has recently been proposed that the AB effect depends on the efficiency of a gating system which facilitates the entrance of relevant input into working memory, while inhibiting irrelevant input. Following the inhibitory response on post T1 distractors, prolonged inhibition of the subsequent T2 is observed. In the present study, we hypothesized that processing facial expressions of emotion would influence this attentional gating. Fearful faces would increase but disgust faces would decrease inhibition of the second target.

Methodology/Principal Findings

We showed that processing fearful versus disgust faces has different effects on these attentional processes. We found that processing fear faces impaired the detection of T2 to a greater extent than did the processing disgust faces. This finding implies emotion-specific modulation of attention.

Conclusions/Significance

Based on the recent literature on attention, our finding suggests that processing fear-related stimuli exerts greater inhibitory responses on distractors relative to processing disgust-related stimuli. This finding is of particular interest for researchers examining the influence of emotional processing on attention and memory in both clinical and normal populations. For example, future research could extend upon the current study to examine whether inhibitory processes invoked by fear-related stimuli may be the mechanism underlying the enhanced learning of fear-related stimuli.

The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

2009-11-19T08:00:00Z

Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis.

Distributed Network, Wireless and Cloud Computing Enabled 3-D Ultrasound; a New Medical Technology Paradigm

2009-11-19T08:00:00Z

Medical technologies are indispensable to modern medicine. However, they have become exceedingly expensive and complex and are not available to the economically disadvantaged majority of the world population in underdeveloped as well as developed parts of the world. For example, according to the World Health Organization about two thirds of the world population does not have access to medical imaging. In this paper we introduce a new medical technology paradigm centered on wireless technology and cloud computing that was designed to overcome the problems of increasing health technology costs. We demonstrate the value of the concept with an example; the design of a wireless, distributed network and central (cloud) computing enabled three-dimensional (3-D) ultrasound system. Specifically, we demonstrate the feasibility of producing a 3-D high end ultrasound scan at a central computing facility using the raw data acquired at the remote patient site with an inexpensive low end ultrasound transducer designed for 2-D, through a mobile device and wireless connection link between them. Producing high-end 3D ultrasound images with simple low-end transducers reduces the cost of imaging by orders of magnitude. It also removes the requirement of having a highly trained imaging expert at the patient site, since the need for hand-eye coordination and the ability to reconstruct a 3-D mental image from 2-D scans, which is a necessity for high quality ultrasound imaging, is eliminated. This could enable relatively untrained medical workers in developing nations to administer imaging and a more accurate diagnosis, effectively saving the lives of people.

Survival and Cardioprotective Benefits of Long-Term Blueberry Enriched Diet in Dilated Cardiomyopathy Following Myocardial Infarction in Rats

2009-11-19T08:00:00Z

Background

Despite remarkable progress in treatment of chronic heart failure (CHF) over the last two decades, mortality, personal suffering and cost remain staggering, and effective interventions are still a challenge. Previously we reported that a blueberry-enriched diet (BD) attenuated necroapoptosis and inflammation in periinfarct area in a rat model of myocardial infarction (MI).

Objectives

To test the hypothesis that BD will attenuate the course of CHF, including mortality and cardiac remodeling during the first year after induction of MI in rats.

Method and Results

Two weeks after coronary artery ligation, rats were divided into two groups of similar average MI size, measured by echocardiography, and then12-mo dietary regimens were initiated as follows: ad libitum regular diet (control, CD, n = 27) and isocaloric food with 2% blueberry supplement (BD, n = 27) also available ad libitum. These dietary groups were compared to each other and to sham group (SH). Mortality over the 12 mo was reduced by 22% in BD compared with CD (p<0.01). In the course of developing CHF, BD had no effect on the body weight, heart rate or blood pressure. Bi-monthly Echo revealed significant attenuation of the LV chamber remodeling, LV posterior wall thinning, and MI expansion in BD compared with CD. In fact, BD arrested the MI expansion.

Conclusion

This is the first experimental evidence that a blueberry-enriched diet has positive effects on the course of CHF and thus warrants consideration for clinical evaluation.

Comparative Analysis of the Global Transcriptome of Anopheles funestus from Mali, West Africa

2009-11-19T08:00:00Z

Background

Anopheles funestus is a principal vector of malaria across much of tropical Africa and is considered one of the most efficient of its kind, yet studies of this species have lagged behind those of its broadly sympatric congener, An. gambiae. In aid of future genomic sequencing of An. funestus, we explored the whole body transcriptome, derived from mixed stage progeny of wild-caught females from Mali, West Africa.

Principal Findings

Here we report the functional annotation and comparative genomics of 2,005 expressed sequence tags (ESTs) from An. funestus, which were assembled with a previous EST set from adult female salivary glands from the same mosquito. The assembled ESTs provided for a nonredundant catalog of 1,035 transcripts excluding mitochondrial sequences.

Conclusions/Significance

Comparison of the An. funestus and An. gambiae transcriptomes using computational and macroarray approaches revealed a high degree of sequence identity despite an estimated 20–80 MY divergence time between lineages. A phylogenetically broader comparative genomic analysis indicated that the most rapidly evolving proteins– those involved in immunity, hematophagy, formation of extracellular structures, and hypothetical conserved proteins– are those that probably play important roles in how mosquitoes adapt to their nutritional and external environments, and therefore could be of greatest interest in disease control.

A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters

2009-11-18T08:00:00Z

Background

Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations.

Methodology/Principal Findings

We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit.

Conclusions/Significance

This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.

Breaking the News or Fueling the Epidemic? Temporal Association between News Media Report Volume and Opioid-Related Mortality

2009-11-18T08:00:00Z

Background

Historical studies of news media have suggested an association between reporting and increased drug abuse. Period effects for substance use have been documented for different classes of legal and illicit substances, with the suspicion that media publicity may have played major roles in their emergence. Previous analyses have drawn primarily from qualitative evidence; the temporal relationship between media reporting volume and adverse health consequences has not been quantified nationally. We set out to explore whether we could find a quantitative relationship between media reports about prescription opioid abuse and overdose mortality associated with these drugs. We assessed whether increases in news media reports occurred before or after increases in overdose deaths.

Methodology/Principal Findings

Our ecological study compared a monthly time series of unintentional poisoning deaths involving short-acting prescription opioid substances, from 1999 to 2005 using multiple cause-of-death data published by the National Center for Health Statistics, to monthly counts of English-language news articles mentioning generic and branded names of prescription opioids obtained from Google News Archives from 1999 to 2005. We estimated the association between media volume and mortality rates by time-lagged regression analyses. There were 24,272 articles and 30,916 deaths involving prescription opioids during the seven-year study period. Nationally, the number of articles mentioning prescription opioids increased dramatically starting in early 2001, following prominent coverage about the nonmedical use of OxyContin. We found a significant association between news reports and deaths, with media reporting preceding fatal opioid poisonings by two to six months and explaining 88% (p<0.0001, df 78) of the variation in mortality.

Conclusions/Significance

While availability, structural, and individual predispositions are key factors influencing substance use, news reporting may enhance the popularity of psychoactive substances. Albeit ecological in nature, our finding suggests the need for further evaluation of the influence of news media on health. Reporting on prescription opioids conforms to historical patterns of news reporting on other psychoactive substances.

SMF-1, SMF-2 and SMF-3 DMT1 Orthologues Regulate and Are Regulated Differentially by Manganese Levels in C. elegans

2009-11-18T08:00:00Z

Manganese (Mn) is an essential metal that can exert toxic effects at high concentrations, eventually leading to Parkinsonism. A major transporter of Mn in mammals is the divalent-metal transporter (DMT1). We characterize here DMT1-like proteins in the nematode C. elegans, which regulate and are regulated by Mn and iron (Fe) content. We identified three new DMT1-like genes in C. elegans: smf-1, smf-2 and smf-3. All three can functionally substitute for loss of their yeast orthologues in S. cerevisiae. In the worm, deletion of smf-1 or smf-3 led to an increased Mn tolerance, while loss of smf-2 led to increased Mn sensitivity. smf mRNA levels measured by QRT-PCR were up-regulated upon low Mn and down-regulated upon high Mn exposures. Translational GFP-fusions revealed that SMF-1 and SMF-3 strongly localize to partially overlapping apical regions of the gut epithelium, suggesting a differential role for SMF-1 and SMF-3 in Mn nutritional intake. Conversely, SMF-2 was detected in the marginal pharyngeal epithelium, possibly involved in metal-sensing. Analysis of metal content upon Mn exposure in smf mutants revealed that SMF-3 is required for normal Mn uptake, while smf-1 was dispensable. Higher smf-2 mRNA levels correlated with higher Fe content, supporting a role for SMF-2 in Fe uptake. In smf-1 and smf-3 but not in smf-2 mutants, increased Mn exposure led to decreased Fe levels, suggesting that both metals compete for transport by SMF-2. Finally, SMF-3 was post-translationally and reversibly down-regulated following Mn-exposure. In sum, we unraveled a complex interplay of transcriptional and post-translational regulations of 3 DMT1-like transporters in two adjacent tissues, which regulate metal-content in C. elegans.

A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

2009-11-18T08:00:00Z

Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis.

Rhesus Monkeys' Valuation of Vocalizations during a Free-Choice Task

2009-11-18T08:00:00Z

Adaptive behavior requires that animals integrate current and past information with their decision-making. One important type of information is auditory-communication signals (i.e., species-specific vocalizations). Here, we tested how rhesus monkeys incorporate the opportunity to listen to different species-specific vocalizations into their decision-making processes. In particular, we tested how monkeys value these vocalizations relative to the opportunity to get a juice reward. To test this hypothesis, monkeys chose one of two targets to get a varying juice reward; at one of those targets, in addition to the juice reward, a vocalization was presented. By titrating the juice amounts at the two targets, we quantified the relationship between the monkeys' juice choices relative to the opportunity to listen to a vocalization. We found that, rhesus were not willing to give up a large juice reward to listen to vocalizations indicating that, relative to a juice reward, listening to vocalizations has a low value.

Mutations in H5N1 Influenza Virus Hemagglutinin that Confer Binding to Human Tracheal Airway Epithelium

2009-11-18T08:00:00Z

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary “dead end.” We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.

The Typical Flight Performance of Blowflies: Measuring the Normal Performance Envelope of Calliphora vicina Using a Novel Corner-Cube Arena

2009-11-18T08:00:00Z

Despite a wealth of evidence demonstrating extraordinary maximal performance, little is known about the routine flight performance of insects. We present a set of techniques for benchmarking performance characteristics of insects in free flight, demonstrated using a model species, and comment on the significance of the performance observed. Free-flying blowflies (Calliphora vicina) were filmed inside a novel mirrored arena comprising a large (1.6 m1.6 m1.6 m) corner-cube reflector using a single high-speed digital video camera (250 or 500 fps). This arrangement permitted accurate reconstruction of the flies' 3-dimensional trajectories without the need for synchronisation hardware, by virtue of the multiple reflections of a subject within the arena. Image sequences were analysed using custom-written automated tracking software, and processed using a self-calibrating bundle adjustment procedure to determine the subject's instantaneous 3-dimensional position. We illustrate our method by using these trajectory data to benchmark the routine flight performance envelope of our flies. Flight speeds were most commonly observed between 1.2 ms⁻¹ and 2.3 ms⁻¹, with a maximum of 2.5 ms⁻¹. Our flies tended to dive faster than they climbed, with a maximum descent rate (−2.4 ms⁻¹) almost double the maximum climb rate (1.2 ms⁻¹). Modal turn rate was around 240°s⁻¹, with maximal rates in excess of 1700°s⁻¹. We used the maximal flight performance we observed during normal flight to construct notional physical limits on the blowfly flight envelope, and used the distribution of observations within that notional envelope to postulate behavioural preferences or physiological and anatomical constraints. The flight trajectories we recorded were never steady: rather they were constantly accelerating or decelerating, with maximum tangential accelerations and maximum centripetal accelerations on the order of 3 g.

In-Group Conformity Sustains Different Foraging Traditions in Capuchin Monkeys (Cebus apella)

2009-11-18T08:00:00Z

Background

Decades of research have revealed rich cultural repertoires encompassing multiple traditions in wild great apes, a picture crucially complemented by experimental simulations with captive apes. Studies with wild capuchin monkeys, the most encephalized simian species, have indicated a New World convergence on these cultural phenomena, involving multiple traditions and tool use. However, experimental studies to date are in conflict with such findings in concluding that capuchins, like other monkeys, show minimal capacities for social learning.

Methodology/Principal Findings

Here we report a new experimental approach in which the alpha male of each of two groups of capuchins was trained to open an artificial foraging device in a quite different way, using either a slide or lift action, then reunited with his group. In each group a majority of monkeys, 8 of 11 and 13 of 14, subsequently mastered the task. Seventeen of the successful 21 monkeys discovered the alternative action to that seeded in the group, performing it a median of 4 times. Nevertheless, all 21 primarily adopted the technique seeded by their group's alpha male. Median proportions of slide versus lift were 0.96 for the group seeded with slide versus 0. 01 for the group seeded with lift.

Conclusions/Significance

These results suggest a striking effect of social conformity in learned behavioral techniques, consistent with field reports of capuchin traditions and convergent on the only other species in which such cultural phenomena have been reported, chimpanzees and humans.

The Real maccoyii: Identifying Tuna Sushi with DNA Barcodes – Contrasting Characteristic Attributes and Genetic Distances

2009-11-18T08:00:00Z

Background

The use of DNA barcodes for the identification of described species is one of the least controversial and most promising applications of barcoding. There is no consensus, however, as to what constitutes an appropriate identification standard and most barcoding efforts simply attempt to pair a query sequence with reference sequences and deem identification successful if it falls within the bounds of some pre-established cutoffs using genetic distance. Since the Renaissance, however, most biological classification schemes have relied on the use of diagnostic characters to identify and place species.

Methodology/Principal Findings

Here we developed a cytochrome c oxidase subunit I character-based key for the identification of all tuna species of the genus Thunnus, and compared its performance with distance-based measures for identification of 68 samples of tuna sushi purchased from 31 restaurants in Manhattan (New York City) and Denver, Colorado. Both the character-based key and GenBank BLAST successfully identified 100% of the tuna samples, while the Barcode of Life Database (BOLD) as well as genetic distance thresholds, and neighbor-joining phylogenetic tree building performed poorly in terms of species identification. A piece of tuna sushi has the potential to be an endangered species, a fraud, or a health hazard. All three of these cases were uncovered in this study. Nineteen restaurant establishments were unable to clarify or misrepresented what species they sold. Five out of nine samples sold as a variant of “white tuna” were not albacore (T. alalunga), but escolar (Lepidocybium flavorunneum), a gempylid species banned for sale in Italy and Japan due to health concerns. Nineteen samples were northern bluefin tuna (T. thynnus) or the critically endangered southern bluefin tuna (T. maccoyii), though nine restaurants that sold these species did not state these species on their menus.

Conclusions/Significance

The Convention on International Trade Endangered Species (CITES) requires that listed species must be identifiable in trade. This research fulfills this requirement for tuna, and supports the nomination of northern bluefin tuna for CITES listing in 2010.

Nestedness of Ectoparasite-Vertebrate Host Networks

2009-11-18T08:00:00Z

Determining the structure of ectoparasite-host networks will enable disease ecologists to better understand and predict the spread of vector-borne diseases. If these networks have consistent properties, then studying the structure of well-understood networks could lead to extrapolation of these properties to others, including those that support emerging pathogens. Borrowing a quantitative measure of network structure from studies of mutualistic relationships between plants and their pollinators, we analyzed 29 ectoparasite-vertebrate host networks—including three derived from molecular bloodmeal analysis of mosquito feeding patterns—using measures of nestedness to identify non-random interactions among species. We found significant nestedness in ectoparasite-vertebrate host lists for habitats ranging from tropical rainforests to polar environments. These networks showed non-random patterns of nesting, and did not differ significantly from published estimates of nestedness from mutualistic networks. Mutualistic and antagonistic networks appear to be organized similarly, with generalized ectoparasites interacting with hosts that attract many ectoparasites and more specialized ectoparasites usually interacting with these same “generalized” hosts. This finding has implications for understanding the network dynamics of vector-born pathogens. We suggest that nestedness (rather than random ectoparasite-host associations) can allow rapid transfer of pathogens throughout a network, and expand upon such concepts as the dilution effect, bridge vectors, and host switching in the context of nested ectoparasite-vertebrate host networks.

Evidence for a Lack of a Direct Transcriptional Suppression of the Iron Regulatory Peptide Hepcidin by Hypoxia-Inducible Factors

2009-11-18T08:00:00Z

Background

Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation.

Methodology/Principal Findings

Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1α or HIF-2α knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased.

Conclusions/Significance

Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

2009-11-18T08:00:00Z

A clinically-related animal model of Parkinson's disease (PD) may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as dopamine (DA) neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. To overcome these limitations, we developed a rat model by stereotaxically (ST) infusing small doses of the mitochondrial complex-I inhibitor, rotenone, into two brain sites: the right ventral tegmental area and the substantia nigra. Four weeks after ST rotenone administration, tyrosine hydroxylase (TH) immunoreactivity in the infusion side decreased by 43.7%, in contrast to a 75.8% decrease observed in rats treated systemically with rotenone (SYS). The rotenone infusion also reduced the DA content, the glutathione and superoxide dismutase activities, and induced alpha-synuclein expression, when compared to the contralateral side. This ST model displays neither peripheral toxicity or mortality and has a high success rate. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD research.

The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

2009-11-18T08:00:00Z

The differentiation and senescence programs of metazoans play key roles in regulating normal development and preventing aberrant cell proliferation, such as cancer. These programs are intimately associated with both the mitotic and apoptotic pathways. Caspase-8 is an apical apoptotic initiator that has recently been appreciated to coordinate non-apoptotic roles in the cell. Most of these functions are attributed to the catalytic domain, however, the amino-terminal death effector domains (DED)s, which belong to the death domain superfamily of proteins, can also play key roles during development. Here we describe a novel role for caspase-8 DEDs in regulating cell differentiation and senescence. Caspase-8 DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial and myeloid cells; genetic deletion or shRNA suppression of caspase-8 disrupts cell differentiation, while re-expression of DEDs rescues this phenotype. Among caspase-8 deficient neuroblastoma cells, DED expression attenuated tumor growth in vivo and proliferation in vitro via disruption of mitosis and cytokinesis, resulting in upregulation of p53 and induction of differentiation markers. These events occur independent of caspase-8 catalytic activity, but require a critical lysine (K156) in a microtubule-binding motif in the second DED domain. The results demonstrate a new function for the DEDs of caspase-8, and describe an unexpected mechanism that contributes to cell differentiation and senescence.

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

2009-11-18T08:00:00Z

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc_con, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

2009-11-18T08:00:00Z

Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

The Transposon Galileo Generates Natural Chromosomal Inversions in Drosophila by Ectopic Recombination

2009-11-18T08:00:00Z

Background

Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii.

Methodology/Principal Findings

To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z³. In the non inverted chromosome, the 2z³ distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric.

Conclusions/Significance

Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity.

Wheat Domestication Accelerated Evolution and Triggered Positive Selection in the β-Xylosidase Enzyme of Mycosphaerella graminicola

2009-11-18T08:00:00Z

Plant cell wall degrading enzymes (PCWDEs) of plant pathogens are receiving increasing interest for their potential to trigger plant defense reactions. In an antagonistic co-evolutionary arms race between host and pathogen, PCWDEs could be under strong selection. Here, we tested the hypothesis that PCWDEs in the fungal wheat pathogen Mycosphaerella graminicola have been positively selected by analyzing ratios of non-synonymous and synonymous nucleotide changes in the genes encoding these enzymes. Analyses of five PCWDEs demonstrated that one (β-xylosidase) has been under strong positive selection and experienced an accelerated rate of evolution. In contrast, PCWDEs in the closest relatives of M. graminicola collected from wild grasses did not show evidence for selection or deviation from a molecular clock. Since the genealogical divergence of M. graminicola from these latter species coincided with the onset of agriculture, we hypothesize that the recent domestication of the host plant and/or agricultural practices triggered positive selection in β-xylosidase and that this enzyme played a key role in the emergence of a host-specialized pathogen.

Rapid Activation of Rac GTPase in Living Cells by Force Is Independent of Src

2009-11-18T08:00:00Z

It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET) method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor) and YPet (a variant yellow fluorescent protein as an acceptor) of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms) of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF) induced Rac activation at a much later time (>30 sec). There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17) was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src activation. These findings suggest that signaling pathways of mechanical forces via integrins might be fundamentally different from those of growth factors.

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

2009-11-18T08:00:00Z

Background

Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting.

Methodology

We developed and implemented an optimized mutation profiling platform (“OncoMap”) to interrogate ~400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact.

Conclusions

Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of “actionable” cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.

Genetic Variation and Recent Positive Selection in Worldwide Human Populations: Evidence from Nearly 1 Million SNPs

2009-11-18T08:00:00Z

Background

Genome-wide scans of hundreds of thousands of single-nucleotide polymorphisms (SNPs) have resulted in the identification of new susceptibility variants to common diseases and are providing new insights into the genetic structure and relationships of human populations. Moreover, genome-wide data can be used to search for signals of recent positive selection, thereby providing new insights into the genetic adaptations that occurred as modern humans spread out of Africa and around the world.

Methodology

We genotyped approximately 500,000 SNPs in 255 individuals (5 individuals from each of 51 worldwide populations) from the Human Genome Diversity Panel (HGDP-CEPH). When merged with non-overlapping SNPs typed previously in 250 of these same individuals, the resulting data consist of over 950,000 SNPs. We then analyzed the genetic relationships and ancestry of individuals without assigning them to populations, and we also identified candidate regions of recent positive selection at both the population and regional (continental) level.

Conclusions

Our analyses both confirm and extend previous studies; in particular, we highlight the impact of various dispersals, and the role of substructure in Africa, on human genetic diversity. We also identified several novel candidate regions for recent positive selection, and a gene ontology (GO) analysis identified several GO groups that were significantly enriched for such candidate genes, including immunity and defense related genes, sensory perception genes, membrane proteins, signal receptors, lipid binding/metabolism genes, and genes involved in the nervous system. Among the novel candidate genes identified are two genes involved in the thyroid hormone pathway that show signals of selection in African Pygmies that may be related to their short stature.

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

2009-11-18T08:00:00Z

CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.

Three Distinct Domains Contribute to Nuclear Transport of Murine Foxp3

2009-11-18T08:00:00Z

Foxp3, a 47-kDa transcription factor, is necessary for the function of CD4+CD25+ regulatory T cells (Tregs), with an essential role in the control of self-reactive T cells and in preventing autoimmunity. Activation of Tregs by TCR engagement results in upregulation of Foxp3 expression, followed by its rapid nuclear transport and binding to chromatin. Here, we identify three distinct Foxp3 domains that contribute to nuclear transport. The first domain (Domain 1) comprises the C-terminal 12 amino acids. The second domain (Domain 2) is located immediately N-terminal to the forkhead domain (FHD), recently reported to be a binding site for the runt-related transcription factor 1/acute myeloid leukemia 1 (Runx1/AML1). The third domain (Domain 3) is located within the N-terminal first 51 amino acids. Unlike the known nuclear localization signals (NLSs), none of these three regions are rich in basic residues and do not bear any similarity to known monopartite or bipartite NLSs that have one or more clusters of basic amino acids. The basic arginine-lysine-lysine-arginine (RKKR) sequence, located 12-aa from the C-terminal end of Foxp3 was previously reported to be a nuclear localization signal (NLS) for several proteins, including for a GFP-Foxp3 hybrid. Evidence is provided here that in the full-length native Foxp3 RKKR does not function as an NLS. The data reported in this study indicates that Foxp3 achieves nuclear transport by binding to other nuclear factors and co-transporting with them to the nucleus.

Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β_42/40 Ratio

2009-11-18T08:00:00Z

Background

Presenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.

Methodology/Principal Findings

To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ₄₂ production are associated with a consistent conformational change in PS1.

Conclusions/Significance

These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ_42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ₄₂ generation, which could be exploited therapeutically.

Novel Protein-Protein Interactions Inferred from Literature Context

2009-11-18T08:00:00Z

We have developed a method that predicts Protein-Protein Interactions (PPIs) based on the similarity of the context in which proteins appear in literature. This method outperforms previously developed PPI prediction algorithms that rely on the conjunction of two protein names in MEDLINE abstracts. We show significant increases in coverage (76% versus 32%) and sensitivity (66% versus 41% at a specificity of 95%) for the prediction of PPIs currently archived in 6 PPI databases. A retrospective analysis shows that PPIs can efficiently be predicted before they enter PPI databases and before their interaction is explicitly described in the literature. The practical value of the method for discovery of novel PPIs is illustrated by the experimental confirmation of the inferred physical interaction between CAPN3 and PARVB, which was based on frequent co-occurrence of both proteins with concepts like Z-disc, dysferlin, and alpha-actinin. The relationships between proteins predicted by our method are broader than PPIs, and include proteins in the same complex or pathway. Dependent on the type of relationships deemed useful, the precision of our method can be as high as 90%. The full set of predicted interactions is available in a downloadable matrix and through the webtool Nermal, which lists the most likely interaction partners for a given protein. Our framework can be used for prioritizing potential interaction partners, hitherto undiscovered, for follow-up studies and to aid the generation of accurate protein interaction maps.

Extrapair Paternity and Maternity in the Three-Toed Woodpecker, Picoides tridactylus: Insights from Microsatellite-Based Parentage Analysis

2009-11-18T08:00:00Z

Molecular techniques have revealed that avian mating systems are more diverse and complex than previously thought. We used microsatellite markers to determine genetic parentage, the prevalence of extrapair paternity and quasi-parasitism (i.e. situations where a male's extrapair mate lay in his nest) in a socially monogamous population of three-toed woodpeckers (Picoides tridactylus) in southern Finland. A total of 129 adults and nestlings, representing 5–9 families annually from 2004–2007, were genotyped at up to ten microsatellite loci. The results of genetic assignment tests confirmed that monogamous parentage characterized the majority (84.6%, 22/26) of broods, and that most (93.8%, 75/80) nestlings were the offspring of their social parents. Two of 80 nestlings (2.5%) in two of 26 broods (7.7%) were sired by extrapair males and quasi-parasitism occurred in 3.8% (3/80) of nestlings and 7.7% (2/26) of broods. Hence, the levels of extrapair parentage were low, possibly because both genetic polygyny and polyandry are constrained by the high paternal effort required for parental care. The co-occurrence of low levels of extrapair paternity and quasi-parasitism are discussed in light of ecological and behavioural factors characterizing the species biology.

Generation and Initial Characterization of FDD Knock In Mice

2009-11-18T08:00:00Z

Background

Mutations in the integral membrane protein 2B [1], also known as BRI₂ [2], a type II trans-membrane domain protein cause two autosomal dominant neurodegenerative diseases, Familial British and Danish Dementia [3]. In these conditions, accumulation of a C-terminal peptide (ABri and ADan) cleaved off from the mutated precursor protein by the pro-protein convertase furin [4], leads to amyloid deposition in the walls of blood vessels and parenchyma of the brain. Recent advances in the understanding of the generation of amyloid in Alzheimer's disease has lead to the finding that BRI₂ interacts with the Amyloid Precursor Protein (APP), decreasing the efficiency of APP processing to generate Aβ [5], [6], [7]. The interaction between the two precursors, APP and BRI₂, and possibly between Aβ and ABri or ADan, could be important in influencing the rate of amyloid production or the tendency of these peptides to aggregate.

Methodology/Principal Findings

We have generated the first BRI₂ Danish Knock-In (FDD_KI) murine model of FDD, expressing the pathogenic decamer duplication in exon 6 of the BRI₂ gene. FDD_KI mice do not show any evident abnormal phenotype, with normal brain histology and no detectable amyloid deposition in blood vessel walls or parenchyma.

Conclusions/Significance

This new murine mouse model will be important to further understand the interaction between APP and BRI₂, and to provide insights into the molecular basis of FDD.

High Mitochondrial DNA Stability in B-Cell Chronic Lymphocytic Leukemia

2009-11-18T08:00:00Z

Background

Chronic Lymphocytic Leukemia (CLL) leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL.

Methodology/Principal Findings

The mitochondrial DNA (mtDNA) control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country). Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II) around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis.

Conclusions/Significance

Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis.

Anthrax Lethal Toxin Induced Lysosomal Membrane Permeabilization and Cytosolic Cathepsin Release Is Nlrp1b/Nalp1b-Dependent

2009-11-18T08:00:00Z

NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or ‘danger signals’. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.

TCF7L2 Polymorphism rs7903146 Is Associated with Coronary Artery Disease Severity and Mortality

2009-11-17T08:00:00Z

Background

TCF7L2 polymorphisms have been consistently associated with type 2 diabetes mellitus in different populations and type 2 diabetes mellitus is a major risk factor for cardiovascular disease, especially coronary artery disease. This study aimed to evaluate the association between TCF7L2 polymorphism rs7903146 and coronary artery disease in diabetic and non-diabetic subjects.

Methods and Results

two populations were studied in order to assess severity of coronary artery disease and cardiovascular events incidence. Eight-hundred and eighty nine subjects who were referred for cardiac catheterization for coronary artery disease diagnosis were cross-sectionally evaluated for coronary lesions (atherosclerotic burden) and 559 subjects from the MASS-II Trial were prospectively followed-up for 5 years and assessed for major cardiovascular events incidence. As expected, rs7903146 T allele was associated with diabetes. Although diabetic patients had a higher prevalence of coronary lesions, no association between TCF7L2 genotype and coronary lesions was found in this subgroup. However, non-diabetic individuals carrying the T allele were associated with a significantly higher frequency of coronary lesions than non-diabetic non-carriers of the risk allele (adjusted OR = 2.32 95%CI 1.27–4.24, p = 0.006). Moreover, presence of multi-vessel coronary artery disease was also associated with the CT or TT genotypes in non-diabetics. Similarly, from the prospective sample analysis, non-diabetics carrying the CT/TT genotypes had significantly more composite cardiovascular end-points events than CC carriers (p = 0.049), mainly due to an increased incidence of death (p = 0.004).

Conclusions

rs7903146 T allele is associated with diabetes and, in non-diabetic individuals, with a higher prevalence and severity of coronary artery disease and cardiovascular events. name of registry site (see list below), registration number, trial registration URL in brackets.

Clinical Trial Registration Information

Medicine, Angioplasty, or Surgery Study (MASS II): Unique identifier: ISRCTN66068876.

Evolutionary Trends of A(H1N1) Influenza Virus Hemagglutinin Since 1918

2009-11-17T08:00:00Z

The Pandemic (H1N1) 2009 is spreading to numerous countries and causing many human deaths. Although the symptoms in humans are mild at present, fears are that further mutations in the virus could lead to a potentially more dangerous outbreak in subsequent months. As the primary immunity-eliciting antigen, hemagglutinin (HA) is the major agent for host-driven antigenic drift in A(H3N2) virus. However, whether and how the evolution of HA is influenced by existing immunity is poorly understood for A(H1N1). Here, by analyzing hundreds of A(H1N1) HA sequences since 1918, we show the first evidence that host selections are indeed present in A(H1N1) HAs. Among a subgroup of human A(H1N1) HAs between 1918~2008, we found strong diversifying (positive) selection at HA₁ 156 and 190. We also analyzed the evolutionary trends at HA₁ 190 and 225 that are critical determinants for receptor-binding specificity of A(H1N1) HA. Different A(H1N1) viruses appeared to favor one of these two sites in host-driven antigenic drift: epidemic A(H1N1) HAs favor HA₁ 190 while the 1918 pandemic and swine HAs favor HA₁ 225. Thus, our results highlight the urgency to understand the interplay between antigenic drift and receptor binding in HA evolution, and provide molecular signatures for monitoring future antigenically drifted 2009 pandemic and seasonal A(H1N1) influenza viruses.

The Evolution of a Capacity to Build Supra-Cellular Ropes Enabled Filamentous Cyanobacteria to Colonize Highly Erodible Substrates

2009-11-17T08:00:00Z

Background

Several motile, filamentous cyanobacteria display the ability to self-assemble into tightly woven or twisted groups of filaments that form macroscopic yarns or ropes, and that are often centimeters long and 50–200 µm in diameter. Traditionally, this trait has been the basis for taxonomic definition of several genera, notably Microcoleus and Hydrocoleum, but the trait has not been associated with any plausible function.

Method and Findings

Through the use of phylogenetic reconstruction, we demonstrate that pedigreed, rope-building cyanobacteria from various habitats do not form a monophyletic group. This is consistent with the hypothesis that rope-building ability was fixed independently in several discrete clades, likely through processes of convergent evolution or lateral transfer. Because rope-building cyanobacteria share the ability to colonize geologically unstable sedimentary substrates, such as subtidal and intertidal marine sediments and non-vegetated soils, it is also likely that this supracellular differentiation capacity imparts a particular fitness advantage in such habitats. The physics of sediment and soil erosion in fact predict that threads in the 50–200 µm size range will attain optimal characteristics to stabilize such substrates on contact.

Conclusions

Rope building is a supracellular morphological adaptation in filamentous cyanobacteria that allows them to colonize physically unstable sedimentary environments, and to act as successful pioneers in the biostabilization process.

The Heterogeneous Allelic Repertoire of Human Toll-Like Receptor (TLR) Genes

2009-11-17T08:00:00Z

Toll-Like Receptors (TLR) are critical elements of the innate arm of the vertebrate immune system. They constitute a multigenic family of receptors which collectively bind a diverse array of – exogeneous as well as endogeneous – ligands. An exponential burst of knowledge has defined their biological role in fight against infections and generation/modulation of auto-immune disorders. Hence, they could at least be conceptually recognized – despite being structurally unrelated – as innate counterparts to Major Histocompatibility Complex (MHC) molecules – equally recognizing antigenic ligands (albeit structurally more homogeneous i.e., peptides), again derived from self and/or non-self sources – preeminent this time in adaptive immunity. Our great disparities in face of infections and/or susceptibility to auto-immune diseases have provoked an intense search for genetic explanations, in part satisfied by the extraordinary MHC allelic repertoire. An equally in-depth and systematic analysis of TLR diversity is lacking despite numerous independent reports of a growing number of SNPs within these loci. The work described here aims at providing a preliminary picture of the allelic repertoire – and not purely SNPs – of all 10 human TLR coding sequences (with exception of TLR3) within a single cohort of up to 100 individuals. It appears from our work that TLR are unequally polymorphic: TLR2 (DNA alleles: 7/protein alleles: 3), 4 (4/3), 7 (6/3), 8 (9/2) and 9 (8/3) being comparatively least diverse whereas TLR1 (11/10), 5 (14/12), 6 (10/8) and 10 (15/10) show a substantial number of alleles. In addition to allelic assignment of a large number of SNPs, 10 new polymorphic positions were hereby identified. Hence this work depicts a first overview of the diversity of almost all human TLR genes, a prelude for large-scale population genetics as well as genetic association studies.

Impact of RTS,S/AS02_A and RTS,S/AS01_B on Genotypes of P. falciparum in Adults Participating in a Malaria Vaccine Clinical Trial

2009-11-17T08:00:00Z

Objective

RTS,S, a candidate vaccine for malaria, is a recombinant protein expressed in yeast containing part of the circumsporozoite protein (CSP) sequence of 3D7 strain of Plasmodium falciparum linked to the hepatitis B surface antigen in a hybrid protein. The RTS,S antigen is formulated with GSK Biologicals' proprietary Adjuvant Systems AS02_A or AS01_B. A recent trial of the RTS,S/AS02_A and RTS,S/AS01_B vaccines evaluated safety, immunogenicity and impact on the development of parasitemia of the two formulations. Parasite isolates from this study were used to determine the molecular impact of RTS,S/AS02_A and RTS,S/AS01_B on the multiplicity of infection (MOI) and the csp allelic characteristics of subsequent parasitemias.

Design

The distribution of csp sequences and the MOI of the infecting strains were examined at baseline and in break-through infections from vaccinated individuals and from those receiving a non-malarial vaccine.

Setting

The study was conducted in Kombewa District, western Kenya.

Participants

Semi-immune adults from the three study arms provided isolates at baseline and during break-through infections.

Outcome

Parasite isolates used for determining MOI and divergence of csp T cell–epitopes were 191 at baseline and 87 from break-through infections.

Results

Grouping recipients of RTS,S/AS01_A and RTS,S/AS02_B together, vaccine recipients identified as parasite-positive by microscopy contained significantly fewer parasite genotypes than recipients of the rabies vaccine comparator (median in pooled RTS,S groups: 3 versus 4 in controls, P = 0.0313). When analyzed separately, parasitaemic individuals in the RTS,S/AS01_B group, but not the RTS,S/AS02_A group, were found to have significantly fewer genotypes than the comparator group. Two individual amino acids found in the vaccine construct (Q339 in Th2R and D371 in Th3R) were observed to differ in incidence between vaccine and comparator groups but in different directions; parasites harboring Q339 were less common among pooled RTS,S/AS vaccine recipients than among recipients of rabies vaccine, whereas parasites with D371 were more common among the RTS,S/AS groups.

Conclusions

It is concluded that both RTS,S/AS vaccines reduce multiplicity of infection. Our results do not support the hypothesis that RTS,S/AS vaccines elicit preferential effects against pfcsp alleles with sequence similarity to the 3D7 pfcsp sequence employed in the vaccine construct.

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

2009-11-17T08:00:00Z

Background

Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis.

Methology/Principal Findings

We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21–66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process.

Conclusions/Significance

Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis.

Immunoproteasome Overexpression Underlies the Pathogenesis of Thyroid Oncocytes and Primary Hypothyroidism: Studies in Humans and Mice

2009-11-17T08:00:00Z

Background

Oncocytes of the thyroid gland (Hürthle cells) are found in tumors and autoimmune diseases. They have a unique appearance characterized by abundant granular eosinophilic cytoplasm and hyperchromatic nucleus. Their pathogenesis has remained, thus far, unknown.

Methodology/Principal Findings

Using transgenic mice chronically expressing IFNγ in thyroid gland, we showed changes in the thyroid follicular epithelium reminiscent of the human oncocyte. Transcriptome analysis comparing transgenic to wild type thyrocytes revealed increased levels of immunoproteasome subunits like LMP2 in transgenics, suggesting an important role of the immunoproteasome in oncocyte pathogenesis. Pharmacologic blockade of the proteasome, in fact, ameliorated the oncocytic phenotype. Genetic deletion of LMP2 subunit prevented the development of the oncocytic phenotype and primary hypothyroidism. LMP2 was also found expressed in oncocytes from patients with Hashimoto thyroiditis and Hürthle cell tumors.

Conclusions/Significance

In summary, we report that oncocytes are the result of an increased immunoproteasome expression secondary to a chronic inflammatory milieu, and suggest LMP2 as a novel therapeutic target for the treatment of oncocytic lesions and autoimmune hypothyroidism.

Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

2009-11-17T08:00:00Z

Background

It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures.

Principal Findings

We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates.

Conclusions/Significance

The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.

Avian Host-Selection by Culex pipiens in Experimental Trials

2009-11-17T08:00:00Z

Evidence from field studies suggests that Culex pipiens, the primary mosquito vector of West Nile virus (WNV) in the northeastern and north central United States, feeds preferentially on American robins (Turdus migratorius). To determine the contribution of innate preferences to observed preference patterns in the field, we conducted host preference trials with a known number of adult female C. pipiens in outdoor cages comparing the relative attractiveness of American robins with two common sympatric bird species, European starling, Sternus vulgaris and house sparrow, Passer domesticus. Host seeking C. pipiens were three times more likely to enter robin-baited traps when with the alternate host was a European starling (n = 4 trials; OR = 3.06; CI [1.42–6.46]) and almost twice more likely when the alternative was a house sparrow (n = 8 trials; OR = 1.80; CI = [1.22–2.90]). There was no difference in the probability of trap entry when two robins were offered (n = 8 trials). Logistic regression analysis determined that the age, sex and weight of the birds, the date of the trial, starting-time, temperature, humidity, wind-speed and age of the mosquitoes had no effect on the probability of a choosing a robin over an alternate bird. Findings indicate that preferential feeding by C. pipiens mosquitoes on certain avian hosts is likely to be inherent, and we discuss the implications innate host preferences may have on enzootic WNV transmission.

Mismatch and G-Stack Modulated Probe Signals on SNP Microarrays

2009-11-17T08:00:00Z

Background

Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. We analyze the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates.

Methodology/Principal Findings

The probe design of GeneChip SNP arrays enables us to disentangle different sources of intensity modulations such as the number of mismatches per duplex, matched and mismatched base pairings including nearest and next-nearest neighbors and their position along the probe sequence. The effect of probe sequence was estimated in terms of triple-motifs with central matches and mismatches which include all 256 combinations of possible base pairings. The probe/target interactions on the chip can be decomposed into nearest neighbor contributions which correlate well with free energy terms of DNA/DNA-interactions in solution. The effect of mismatches is about twice as large as that of canonical pairings. Runs of guanines (G) and the particular type of mismatched pairings formed in cross-allelic probe/target duplexes constitute sources of systematic biases of the probe signals with consequences for genotyping and copy number estimates. The poly-G effect seems to be related to the crowded arrangement of probes which facilitates complex formation of neighboring probes with at minimum three adjacent G's in their sequence.

Conclusions

The applied method of “triple-averaging” represents a model-free approach to estimate the mean intensity contributions of different sequence motifs which can be applied in calibration algorithms to correct signal values for sequence effects. Rules for appropriate sequence corrections are suggested.

Regulatory Function of a Novel Population of Mouse Autoantigen-Specific Foxp3⁻ Regulatory T Cells Depends on IFN-γ, NO, and Contact with Target Cells

2009-11-17T08:00:00Z

Background

Both naturally arising Foxp3⁺ and antigen-induced Foxp3⁻ regulatory T cells (Treg) play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy.

Methodology/Principal Findings

Using a novel class II MHC tetramer, we have isolated a population of CD4⁺ Foxp3⁻ T cells specific for the autoantigen glutamic acid decarboxylase p286–300 peptide (NR286 T cells) from diabetes-resistant non-obese resistant (NOR) mice. These Foxp3⁻ NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-γ was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg.

Conclusions/Significance

These studies identified a unique population of autoantigen-specific Foxp3⁻ Treg that can exert their regulatory function dependent on not only IFN-γ and NO but also cell contact with target cells.

Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans

2009-11-17T08:00:00Z

Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a K_m of approximately 150 µM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans.

Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of Monocyte Migration in Immune-Mediated Inflammatory Disease

2009-11-17T08:00:00Z

Background

Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man.

Methods/Principal Findings

We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (^99mTc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4×10⁻³ (0.95−5.1×10⁻³) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion.

Conclusions/Significance

The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.

Selective Inhibition of Retinal Angiogenesis by Targeting PI3 Kinase

2009-11-17T08:00:00Z

Ocular neovascularisation is a pathological hallmark of some forms of debilitating blindness including diabetic retinopathy, age related macular degeneration and retinopathy of prematurity. Current therapies for delaying unwanted ocular angiogenesis include laser surgery or molecular inhibition of the pro-angiogenic factor VEGF. However, targeting of angiogenic pathways other than, or in combination to VEGF, may lead to more effective and safer inhibitors of intraocular angiogenesis. In a small chemical screen using zebrafish, we identify LY294002 as an effective and selective inhibitor of both developmental and ectopic hyaloid angiogenesis in the eye. LY294002, a PI3 kinase inhibitor, exerts its anti-angiogenic effect in a dose-dependent manner, without perturbing existing vessels. Significantly, LY294002 delivered by intraocular injection, significantly inhibits ocular angiogenesis without systemic side-effects and without diminishing visual function. Thus, targeting of PI3 kinase pathways has the potential to effectively and safely treat neovascularisation in eye disease.

Longitudinal Evaluation of an N-Ethyl-N-Nitrosourea-Created Murine Model with Normal Pressure Hydrocephalus

2009-11-17T08:00:00Z

Background

Normal-pressure hydrocephalus (NPH) is a neurodegenerative disorder that usually occurs late in adult life. Clinically, the cardinal features include gait disturbances, urinary incontinence, and cognitive decline.

Methodology/Principal Findings

Herein we report the characterization of a novel mouse model of NPH (designated p23-ST1), created by N-ethyl-N-nitrosourea (ENU)-induced mutagenesis. The ventricular size in the brain was measured by 3-dimensional micro-magnetic resonance imaging (3D-MRI) and was found to be enlarged. Intracranial pressure was measured and was found to fall within a normal range. A histological assessment and tracer flow study revealed that the cerebral spinal fluid (CSF) pathway of p23-ST1 mice was normal without obstruction. Motor functions were assessed using a rotarod apparatus and a CatWalk gait automatic analyzer. Mutant mice showed poor rotarod performance and gait disturbances. Cognitive function was evaluated using auditory fear-conditioned responses with the mutant displaying both short- and long-term memory deficits. With an increase in urination frequency and volume, the mutant showed features of incontinence. Nissl substance staining and cell-type-specific markers were used to examine the brain pathology. These studies revealed concurrent glial activation and neuronal loss in the periventricular regions of mutant animals. In particular, chronically activated microglia were found in septal areas at a relatively young age, implying that microglial activation might contribute to the pathogenesis of NPH. These defects were transmitted in an autosomal dominant mode with reduced penetrance. Using a whole-genome scan employing 287 single-nucleotide polymorphic (SNP) markers and further refinement using six additional SNP markers and four microsatellite markers, the causative mutation was mapped to a 5.3-cM region on chromosome 4.

Conclusions/Significance

Our results collectively demonstrate that the p23-ST1 mouse is a novel mouse model of human NPH. Clinical observations suggest that dysfunctions and alterations in the brains of patients with NPH might occur much earlier than the appearance of clinical signs. p23-ST1 mice provide a unique opportunity to characterize molecular changes and the pathogenic mechanism of NPH.

Microglia and Microglia-Like Cell Differentiated from DC Inhibit CD4 T Cell Proliferation

2009-11-17T08:00:00Z

The central nervous system (CNS) is generally regarded as a site of immune privilege, whether the antigen presenting cells (APCs) are involved in the immune homeostasis of the CNS is largely unknown. Microglia and DCs are major APCs in physiological and pathological conditions, respectively. In this work, primary microglia and microglia-like cells obtained by co-culturing mature dendritic cells with CNS endothelial cells in vitro were functional evaluated. We found that microglia not only cannot prime CD4 T cells but also inhibit mature DCs (maDCs) initiated CD4 T cells proliferation. More importantly, endothelia from the CNS can differentiate maDCs into microglia-like cells (MLCs), which possess similar phenotype and immune inhibitory function as microglia. Soluble factors including NO lie behind the suppression of CD4 T cell proliferation induced by both microglia and MLCs. All the data indicate that under physiological conditions, microglia play important roles in maintaining immune homeostasis of the CNS, whereas in a pathological situation, the infiltrated DCs can be educated by the local microenvironment and differentiate into MLCs with inhibitory function.

Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro

2009-11-17T08:00:00Z

Background

Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases.

Methodology/Principal Findings

Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment.

Conclusions/Significance

These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.

Dihydroartemisinin-Piperaquine and Artemether-Lumefantrine for Treating Uncomplicated Malaria in African Children: A Randomised, Non-Inferiority Trial

2009-11-17T08:00:00Z

Background

Artemisinin combination therapies (ACTs) are currently the preferred option for treating uncomplicated malaria. Dihydroartemisinin-piperaquine (DHA-PQP) is a promising fixed-dose ACT with limited information on its safety and efficacy in African children.

Methodology/Principal Findings

The non-inferiority of DHA-PQP versus artemether-lumefantrine (AL) in children 6–59 months old with uncomplicated P. falciparum malaria was tested in five African countries (Burkina Faso, Kenya, Mozambique, Uganda and Zambia). Patients were randomised (2∶1) to receive either DHA-PQP or AL. Non-inferiority was assessed using a margin of −5% for the lower limit of the one-sided 97.5% confidence interval on the treatment difference (DHA-PQP vs. AL) of the day 28 polymerase chain reaction (PCR) corrected cure rate. Efficacy analysis was performed in several populations, and two of them are presented here: intention-to-treat (ITT) and enlarged per-protocol (ePP). 1553 children were randomised, 1039 receiving DHA-PQP and 514 AL. The PCR-corrected day 28 cure rate was 90.4% (ITT) and 94.7% (ePP) in the DHA-PQP group, and 90.0% (ITT) and 95.3% (ePP) in the AL group. The lower limits of the one-sided 97.5% CI of the difference between the two treatments were −2.80% and −2.96%, in the ITT and ePP populations, respectively. In the ITT population, the Kaplan-Meier estimate of the proportion of new infections up to Day 42 was 13.55% (95% CI: 11.35%–15.76%) for DHA-PQP vs 24.00% (95% CI: 20.11%–27.88%) for AL (p<0.0001).

Conclusions/Significance

DHA-PQP is as efficacious as AL in treating uncomplicated malaria in African children from different endemicity settings, and shows a comparable safety profile. The occurrence of new infections within the 42-day follow up was significantly lower in the DHA-PQP group, indicating a longer post-treatment prophylactic effect.

Trial Registration

Controlled-trials.com ISRCTN16263443

Control of TCF-4 Expression by VDR and Vitamin D in the Mouse Mammary Gland and Colorectal Cancer Cell Lines

2009-11-17T08:00:00Z

Background

The vitamin D receptor (VDR) pathway is important in the prevention and potentially in the treatment of many cancers. One important mechanism of VDR action is related to its interaction with the Wnt/β-catenin pathway. Agonist-bound VDR inhibits the oncogenic Wnt/β-catenin/TCF pathway by interacting directly with β-catenin and in some cells by increasing cadherin expression which, in turn, recruits β-catenin to the membrane. Here we identify TCF-4, a transcriptional regulator and β-catenin binding partner as an indirect target of the VDR pathway.

Methodology/Principal Findings

In this work, we show that TCF-4 (gene name TCF7L2) is decreased in the mammary gland of the VDR knockout mouse as compared to the wild-type mouse. Furthermore, we show 1,25(OH)₂D₃ increases TCF-4 at the RNA and protein levels in several human colorectal cancer cell lines, the effect of which is completely dependent on the VDR. In silico analysis of the human and mouse TCF7L2 promoters identified several putative VDR binding elements. Although TCF7L2 promoter reporters responded to exogenous VDR, and 1,25(OH)₂D₃, mutation analysis and chromatin immunoprecipitation assays, showed that the increase in TCF7L2 did not require recruitment of the VDR to the identified elements and indicates that the regulation by VDR is indirect. This is further confirmed by the requirement of de novo protein synthesis for this up-regulation.

Conclusions/Significance

Although it is generally assumed that binding of β-catenin to members of the TCF/LEF family is cancer-promoting, recent studies have indicated that TCF-4 functions instead as a transcriptional repressor that restricts breast and colorectal cancer cell growth. Consequently, we conclude that the 1,25(OH)₂D₃/VDR-mediated increase in TCF-4 may have a protective role in colon cancer as well as diabetes and Crohn's disease.

Disruption of Mitochondrial DNA Replication in Drosophila Increases Mitochondrial Fast Axonal Transport In Vivo

2009-11-17T08:00:00Z

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.

Experimental Passage of St. Louis Encephalitis Virus In Vivo in Mosquitoes and Chickens Reveals Evolutionarily Significant Virus Characteristics

2009-11-17T08:00:00Z

St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) was the major cause of epidemic flaviviral encephalitis in the U.S. prior to the introduction of West Nile virus (WNV) in 1999. However, outbreaks of SLEV have been significantly more limited then WNV in terms of levels of activity and geographic dispersal. One possible explanation for these variable levels of activity is that differences in the potential for each virus to adapt to its host cycle exist. The need for arboviruses to replicate in disparate hosts is thought to result in constraints on both evolution and host-specific adaptation. If cycling is the cause of genetic stability observed in nature and arboviruses lack host specialization, then sequential passage should result in both the accumulation of mutations and specialized viruses better suited for replication in that host. Previous studies suggest that WNV and SLEV differ in capacity for both genetic change and host specialization, and in the costs each accrues from specializing. In an attempt to clarify how selective pressures contribute to epidemiological patterns of WNV and SLEV, we evaluated mutant spectra size, consensus genetic change, and phenotypic changes for SLEV in vivo following 20 sequential passages via inoculation in either Culex pipiens mosquitoes or chickens. Results demonstrate that the capacity for genetic change is large for SLEV and that the size of the mutant spectrum is host-dependent using our passage methodology. Despite this, a general lack of consensus change resulted from passage in either host, a result that contrasts with the idea that constraints on evolution in nature result from host cycling alone. Results also suggest that a high level of adaptation to both hosts already exists, despite host cycling. A strain significantly more infectious in chickens did emerge from one lineage of chicken passage, yet other lineages and all mosquito passage strains did not display measurable host-specific fitness gains. In addition, increased infectivity in chickens did not decrease infectivity in mosquitoes, which further contrasts the concept of fitness trade-offs for arboviruses.

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

2009-11-17T08:00:00Z

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.

Transcriptional Regulation and Biological Functions of Selenium-Binding Protein 1 in Colorectal Cancer In Vitro and in Nude Mouse Xenografts

2009-11-16T08:00:00Z

Background

It has been shown that selenium-binding protein 1 (SBP1) is significantly downregulated in different human cancers. Its regulation and function have not yet been established.

Methodology and Principal Findings

We show that the SBP1 promoter is hypermethylated in colon cancer tissues and human colon cancer cells. Treatment with 5′-Aza-2′-deoxycytidine leads to demethylation of the SBP1 promoter and to an increase of SBP1 promoter activity, rescues SBP1 mRNA and protein expression in human colon cancer cells. Additionally, overexpression of SBP1 sensitizes colon cancer cells to H₂O₂-induced apoptosis, inhibits cancer cell migration in vitro and inhibits tumor growth in nude mice.

Conclusion and Significance

These data demonstrate that SBP1 has tumor suppressor functions that are inhibited in colorectal cancer through epigenetic silencing.

A C. elegans Model for Mitochondrial Fatty Acid Synthase II: The Longevity-Associated Gene W09H1.5/mecr-1 Encodes a 2-trans-Enoyl-Thioester Reductase

2009-11-16T08:00:00Z

Our recognition of the mitochondria as being important sites of fatty acid biosynthesis is continuously unfolding, especially in light of new data becoming available on compromised fatty acid synthase type 2 (FASII) in mammals. For example, perturbed regulation of murine 17β-HSD8 encoding a component of the mitochondrial FASII enzyme 3-oxoacyl-thioester reductase is implicated in polycystic kidney disease. In addition, over-expression in mice of the Mecr gene coding for 2-trans-enoyl-thioester reductase, also of mitochondrial FASII, leads to impaired heart function. However, mouse knockouts for mitochondrial FASII have hitherto not been reported and, hence, there is a need to develop alternate metazoan models such as nematodes or fruit flies. Here, the identification of Caenorhabditis elegans W09H1.5/MECR-1 as a 2-trans-enoyl-thioester reductase of mitochondrial FASII is reported. To identify MECR-1, Saccharomyces cerevisiae etr1Δ mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p. These yeast mutants fail to synthesize sufficient levels of lipoic acid or form cytochrome complexes, and cannot respire or grow on non-fermentable carbon sources. A mutant yeast strain ectopically expressing nematode mecr-1 was shown to contain reductase activity and resemble the self-complemented mutant strain for these phenotype characteristics. Since MECR-1 was not intentionally targeted for compartmentalization using a yeast mitochondrial leader sequence, this inferred that the protein represented a physiologically functional mitochondrial 2-trans-enoyl-thioester reductase. In accordance with published findings, RNAi-mediated knockdown of mecr-1 in C. elegans resulted in life span extension, presumably due to mitochondrial dysfunction. Moreover, old mecr-1(RNAi) worms had better internal organ appearance and were more mobile than control worms, indicating a reduced physiological age. This is the first report on RNAi work dedicated specifically to curtailing mitochondrial FASII in metazoans. The availability of affected survivors will help to position C. elegans as an excellent model for future pursuits in the emerging field of mitochondrial FASII research.

Integration of the β-Catenin-Dependent Wnt Pathway with Integrin Signaling through the Adaptor Molecule Grb2

2009-11-16T08:00:00Z

Background

The complexity of wnt signaling likely stems from two sources: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified.

Methodology/Principal Findings

Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of β-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-β-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of β-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK), and this is blocked by DN-Grb2.

Conclusions/Significance

These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling.

Transcriptome Profiling of Human Pre-Implantation Development

2009-11-16T08:00:00Z

Background

Preimplantation development is a crucial step in early human development. However, the molecular basis of human preimplantation development is not well known.

Methodology

By applying microarray on 397 human oocytes and embryos at six developmental stages, we studied the transcription dynamics during human preimplantation development.

Principal Findings

We found that the preimplantation development consisted of two main transitions: from metaphase-II oocyte to 4-cell embryo where mainly the maternal genes were expressed, and from 8-cell embryo to blastocyst with down-regulation of the maternal genes and up-regulation of embryonic genes. Human preimplantation development proved relatively autonomous. Genes predominantly expressed in oocytes and embryos are well conserved during evolution.

Significance

Our database and findings provide fundamental resources for understanding the genetic network controlling early human development.

PLoS ONE Alerts: New Articles

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Heterosubtype Neutralizing Responses to Influenza A (H5N1) Viruses Are Mediated by Antibodies to Virus Haemagglutinin

Akt and SHIP Modulate Francisella Escape from the Phagosome and Induction of the Fas-Mediated Death Pathway

TLR–Dependent Control of Francisella tularensis Infection and Host Inflammatory Responses

A Mechanistic Niche Model for Measuring Species' Distributional Responses to Seasonal Temperature Gradients

Modulation of Outer Hair Cell Electromotility by Cochlear Supporting Cells and Gap Junctions

A Spectrum of Severe Familial Liver Disorders Associate with Telomerase Mutations

Genome-Wide Scan for Signatures of Human Population Differentiation and Their Relationship with Natural Selection, Functional Pathways and Diseases

IL-6 Deficiency Attenuates Murine Diet-Induced Non-Alcoholic Steatohepatitis

Lrp4, a Novel Receptor for Dickkopf 1 and Sclerostin, Is Expressed by Osteoblasts and Regulates Bone Growth and Turnover In Vivo

Early-Onset and Robust Amyloid Pathology in a New Homozygous Mouse Model of Alzheimer's Disease

CRX Is a Diagnostic Marker of Retinal and Pineal Lineage Tumors

Motor and Linguistic Linking of Space and Time in the Cerebellum

p38γ Mitogen-Activated Protein Kinase Is a Key Regulator in Skeletal Muscle Metabolic Adaptation in Mice

Divergent Mechanisms Controlling Hypoxic Sensitivity and Lifespan by the DAF-2/Insulin/IGF-Receptor Pathway

Combination of Real-Value Smell and Metaphor Expression Aids Yeast Detection

Stem Loop Sequences Specific to Transposable Element IS605 Are Found Linked to Lipoprotein Genes in Borrelia Plasmids

Absence of ERRα in Female Mice Confers Resistance to Bone Loss Induced by Age or Estrogen-Deficiency

On Theoretical Models of Gene Expression Evolution with Random Genetic Drift and Natural Selection

Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

Gene Expression Profiling during Early Acute Febrile Stage of Dengue Infection Can Predict the Disease Outcome

Synergy between Repellents and Organophosphates on Bed Nets: Efficacy and Behavioural Response of Natural Free-Flying An. gambiae Mosquitoes

Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Socioeconomic Predictors of Cognition in Ugandan Children: Implications for Community Interventions

Feasibility of Provider-Initiated HIV Testing and Counselling of Tuberculosis Patients Under the TB Control Programme in Two Districts of South India

Inhibition of PI3K-Akt Signaling Blocks Exercise-Mediated Enhancement of Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

The Astronomical Orientation of Ancient Greek Temples

Epigenetic Features of Human Mesenchymal Stem Cells Determine Their Permissiveness for Induction of Relevant Transcriptional Changes by SYT-SSX1

Metabolite Profiling Identifies Candidate Markers Reflecting the Clinical Adaptations Associated with Roux-en-Y Gastric Bypass Surgery

Genome-Wide Analysis of Immune Activation in Human T and B Cells Reveals Distinct Classes of Alternatively Spliced Genes

Performance of an Influenza Rapid Test in Children in a Primary Healthcare Setting in Nicaragua

H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Phenotypic Plasticity of Mouse Spermatogonial Stem Cells

Generation and Characterization of Fmr1 Knockout Zebrafish

Evidence for Anomalous Network Connectivity during Working Memory Encoding in Schizophrenia: An ICA Based Analysis

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

Experimental Diabetes Mellitus Exacerbates Tau Pathology in a Transgenic Mouse Model of Alzheimer's Disease

The Background of Mitochondrial DNA Haplogroup J Increases the Sensitivity of Leber's Hereditary Optic Neuropathy Cells to 2,5-Hexanedione Toxicity

Emotional Modulation of Attention: Fear Increases but Disgust Reduces the Attentional Blink

The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

Distributed Network, Wireless and Cloud Computing Enabled 3-D Ultrasound; a New Medical Technology Paradigm

Survival and Cardioprotective Benefits of Long-Term Blueberry Enriched Diet in Dilated Cardiomyopathy Following Myocardial Infarction in Rats

Comparative Analysis of the Global Transcriptome of Anopheles funestus from Mali, West Africa

A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters

Breaking the News or Fueling the Epidemic? Temporal Association between News Media Report Volume and Opioid-Related Mortality

SMF-1, SMF-2 and SMF-3 DMT1 Orthologues Regulate and Are Regulated Differentially by Manganese Levels in C. elegans

A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

Rhesus Monkeys' Valuation of Vocalizations during a Free-Choice Task

Mutations in H5N1 Influenza Virus Hemagglutinin that Confer Binding to Human Tracheal Airway Epithelium

The Typical Flight Performance of Blowflies: Measuring the Normal Performance Envelope of Calliphora vicina Using a Novel Corner-Cube Arena

In-Group Conformity Sustains Different Foraging Traditions in Capuchin Monkeys (Cebus apella)

The Real maccoyii: Identifying Tuna Sushi with DNA Barcodes – Contrasting Characteristic Attributes and Genetic Distances

Nestedness of Ectoparasite-Vertebrate Host Networks

Evidence for a Lack of a Direct Transcriptional Suppression of the Iron Regulatory Peptide Hepcidin by Hypoxia-Inducible Factors

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

The Transposon Galileo Generates Natural Chromosomal Inversions in Drosophila by Ectopic Recombination

Wheat Domestication Accelerated Evolution and Triggered Positive Selection in the β-Xylosidase Enzyme of Mycosphaerella graminicola

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Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

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Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

Three Distinct Domains Contribute to Nuclear Transport of Murine Foxp3

Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β42/40 Ratio

Novel Protein-Protein Interactions Inferred from Literature Context

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Generation and Initial Characterization of FDD Knock In Mice

High Mitochondrial DNA Stability in B-Cell Chronic Lymphocytic Leukemia

Anthrax Lethal Toxin Induced Lysosomal Membrane Permeabilization and Cytosolic Cathepsin Release Is Nlrp1b/Nalp1b-Dependent

TCF7L2 Polymorphism rs7903146 Is Associated with Coronary Artery Disease Severity and Mortality

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The Evolution of a Capacity to Build Supra-Cellular Ropes Enabled Filamentous Cyanobacteria to Colonize Highly Erodible Substrates

The Heterogeneous Allelic Repertoire of Human Toll-Like Receptor (TLR) Genes

Impact of RTS,S/AS02A and RTS,S/AS01B on Genotypes of P. falciparum in Adults Participating in a Malaria Vaccine Clinical Trial

Catastrophic NAD⁺ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β_42/40 Ratio

Impact of RTS,S/AS02_A and RTS,S/AS01_B on Genotypes of P. falciparum in Adults Participating in a Malaria Vaccine Clinical Trial

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