Detection of regulator genes and eQTLs in gene networks

Genetic differences between individuals associated to quantitative phenotypic traits, including disease states, are usually found in non-coding genomic regions. These genetic variants are often also associated to differences in expression levels of nearby genes (they are "expression quantitative trait loci" or eQTLs for short) and presumably play a gene regulatory role, affecting the status of molecular networks of interacting genes, proteins and metabolites. Computational systems biology approaches to reconstruct causal gene networks from large-scale omics data have therefore become essential to understand the structure of networks controlled by eQTLs together with other regulatory genes, and to generate detailed hypotheses about the molecular mechanisms that lead from genotype to phenotype. Here we review the main analytical methods and softwares to identify eQTLs and their associated genes, to reconstruct co-expression networks and modules, to reconstruct causal Bayesian gene and module networks, and to validate predicted networks in silico.Comment: minor revision with typos corrected; review article; 24 pages, 2 figure

NMDA Receptors on Non-Dopaminergic Neurons in the VTA Support Cocaine Sensitization

The initiation of behavioral sensitization to cocaine and other psychomotor stimulants is thought to reflect N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic plasticity in the mesolimbic dopamine (DA) circuitry. The importance of drug induced NMDAR mediated adaptations in ventral tegmental area (VTA) DA neurons, and its association with drug seeking behaviors, has recently been evaluated in Cre-loxp mice lacking functional NMDARs in DA neurons expressing Cre recombinase under the control of the endogenous dopamine transporter gene (NR1(DATCre) mice).Using an additional NR1(DATCre) mouse transgenic model, we demonstrate that while the selective inactivation of NMDARs in DA neurons eliminates the induction of molecular changes leading to synaptic strengthening, behavioral measures such as cocaine induced locomotor sensitization and conditioned place preference remain intact in NR1(DATCre) mice. Since VTA DA neurons projecting to the prefrontal cortex and amygdala express little or no detectable levels of the dopamine transporter, it has been speculated that NMDA receptors in DA neurons projecting to these brain areas may have been spared in NR1(DATCre) mice. Here we demonstrate that the NMDA receptor gene is ablated in the majority of VTA DA neurons, including those exhibiting undetectable DAT expression levels in our NR1(DATCre) transgenic model, and that application of an NMDAR antagonist within the VTA of NR1(DATCre) animals still blocks sensitization to cocaine.These results eliminate the possibility of NMDAR mediated neuroplasticity in the different DA neuronal subpopulations in our NR1(DATCre) mouse model and therefore suggest that NMDARs on non-DA neurons within the VTA must play a major role in cocaine-related addictive behavior

Impairment of the Plasmodium falciparum Erythrocytic Cycle Induced by Angiotensin Peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1–7). Parasite infection decreased Ang-(1–7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1–7) decreased the level of infection in an A779 (specific antagonist of Ang-(1–7) receptor, MAS)-sensitive manner. 10−7 M PD123319, an AT2 receptor antagonist, partially reversed the effects of Ang-(1–7) and Ang II. However, 10−6 M losartan, an antagonist of the AT1 receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10−8 M Ang II or 10−8 M Ang-(1–7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10−7 M A779. 10−6 M dibutyryl-cAMP increased the level of infection and 10−7 M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1–7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1–7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus

Impairment of the Plasmodium falciparum Erythrocytic Cycle Induced by Angiotensin Peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1–7). Parasite infection decreased Ang-(1–7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1–7) decreased the level of infection in an A779 (specific antagonist of Ang-(1–7) receptor, MAS)-sensitive manner. 10−7 M PD123319, an AT2 receptor antagonist, partially reversed the effects of Ang-(1–7) and Ang II. However, 10−6 M losartan, an antagonist of the AT1 receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10−8 M Ang II or 10−8 M Ang-(1–7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10−7 M A779. 10−6 M dibutyryl-cAMP increased the level of infection and 10−7 M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1–7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1–7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus

First experiences in the implementation of biometric technology to link data from Health and Demographic Surveillance Systems with health facility data

BACKGROUND: In developing countries, Health and Demographic Surveillance Systems (HDSSs) provide a framework for tracking demographic and health dynamics over time in a defined geographical area. Many HDSSs co-exist with facility-based data sources in the form of Health Management Information Systems (HMIS). Integrating both data sources through reliable record linkage could provide both numerator and denominator populations to estimate disease prevalence and incidence rates in the population and enable determination of accurate health service coverage. OBJECTIVE: To measure the acceptability and performance of fingerprint biometrics to identify individuals in demographic surveillance populations and those attending health care facilities serving the surveillance populations. METHODOLOGY: Two HDSS sites used fingerprint biometrics for patient and/or surveillance population participant identification. The proportion of individuals for whom a fingerprint could be successfully enrolled were characterised in terms of age and sex. RESULTS: Adult (18-65 years) fingerprint enrolment rates varied between 94.1% (95% CI 93.6-94.5) for facility-based fingerprint data collection at the Africa Centre site to 96.7% (95% CI 95.9-97.6) for population-based fingerprint data collection at the Agincourt site. Fingerprint enrolment rates in children under 1 year old (Africa Centre site) were only 55.1% (95% CI 52.7-57.4). By age 5, child fingerprint enrolment rates were comparable to those of adults. CONCLUSION: This work demonstrates the feasibility of fingerprint-based individual identification for population-based research in developing countries. Record linkage between demographic surveillance population databases and health care facility data based on biometric identification systems would allow for a more comprehensive evaluation of population health, including the ability to study health service utilisation from a population perspective, rather than the more restrictive health service perspective

Anopheles Gambiae PRS1 Modulates Plasmodium Development at Both Midgut and Salivary Gland Steps

Background: Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches. Methodology/Principal Findings: PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues. Conclusions/Significance: While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history

Genome-Wide Analysis Reveals a Complex Pattern of Genomic Imprinting in Mice

Parent-of-origin–dependent gene expression resulting from genomic imprinting plays an important role in modulating complex traits ranging from developmental processes to cognitive abilities and associated disorders. However, while gene-targeting techniques have allowed for the identification of imprinted loci, very little is known about the contribution of imprinting to quantitative variation in complex traits. Most studies, furthermore, assume a simple pattern of imprinting, resulting in either paternal or maternal gene expression; yet, more complex patterns of effects also exist. As a result, the distribution and number of different imprinting patterns across the genome remain largely unexplored. We address these unresolved issues using a genome-wide scan for imprinted quantitative trait loci (iQTL) affecting body weight and growth in mice using a novel three-generation design. We identified ten iQTL that display much more complex and diverse effect patterns than previously assumed, including four loci with effects similar to the callipyge mutation found in sheep. Three loci display a new phenotypic pattern that we refer to as bipolar dominance, where the two heterozygotes are different from each other while the two homozygotes are identical to each other. Our study furthermore detected a paternally expressed iQTL on Chromosome 7 in a region containing a known imprinting cluster with many paternally expressed genes. Surprisingly, the effects of the iQTL were mostly restricted to traits expressed after weaning. Our results imply that the quantitative effects of an imprinted allele at a locus depend both on its parent of origin and the allele it is paired with. Our findings also show that the imprinting pattern of a locus can be variable over ontogenetic time and, in contrast to current views, may often be stronger at later stages in life

Normalisation to Blood Activity Is Required for the Accurate Quantification of Na/I Symporter Ectopic Expression by SPECT/CT in Individual Subjects

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used 99mTc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to ‘noisy’, and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy

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

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. © 2009 Kam et al.published_or_final_versio