Rpe65-Gene Transfer Using an Integration-Deficient Lentiviral Vector

Purpose: We previously demonstrated efficient retinal rescue of RPE65 mouse models (Rpe65-/- (Bemelmans et al, 2006) and Rpe65R91W/R91W mice) using a HIV1-derived lentiviral vector encoding for the mouse RPE65 cDNA. In order to optimize a lentiviral vector as an alternative tool for RPE65-derived Leber Congenital Amaurosis clinical trials, we evaluated the efficiency of an integration-deficient lentiviral vector (IDLV) encoding the human RPE65 cDNA to restore retinal function in the Rpe65R91W/R91W mice. Methods: An HIV-1-derived lentiviral vector expressing either the hrGFPII or the human Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced by transient transfection of 293T cells. A LQ-integrase mutant was used to generate the IDLV vectors. IDLV-R0.8-hRPE65 or hrGFPII were injected subretinally into 1 month-old Rpe65R91W/R91W mice. Functional rescue was assessed by ERG (1 and 3 months post-injection) and cone survival by immunohistology. Results: An increased light sensitivity was detected by scotopic ERG in animals injected with IDLV-R0.8-hRPE65 compared to hrGFPII-treated animals or untreated mice. However the improvement was delayed compared to integration-proficient LV and observed at 3 months but not 1 month post-injection. Immunolabelling of cone markers showed an increased number of cones in the transduced area compared to control groups. Conclusions: The IDLV-R0.8-hRPE65 vectors allow retinal improvement in the Rpe65R91W/R91W mice. Both rod function and cone survival were demonstrated even if there is a delay in the rescue as assessed by scotopic ERG. Integration-deficient vectors minimize insertional mutagenesis and thus are safer candidates for human application. Further experiments using large animals are now needed to validate correct gene transfer and expression of the RPE65 gene as well as tolerance of the vector after subretinal injection before envisaging a clinical trial application.

A gain-of-function allele of TPC1 activates oxylipin biogenesis after leaf wounding in Arabidopsis.

Jasmonates, potent lipid mediators of defense gene expression in plants, are rapidly synthesized in response to wounding. These lipid mediators also stimulate their own production via a positive feedback circuit, which depends on both JA synthesis and JA signaling. To date, molecular components regulating the activation of jasmonate biogenesis and its feedback loop have been poorly characterized. We employed a genetic screen capable of detecting the misregulated activity of 13-lipoxygenase, which operates at the entry point of the jasmonate biosynthesis pathway. Leaf extracts from the Arabidopsis fou2 (fatty acid oxygenation upregulated 2) mutant displayed an increased capacity to catalyze the synthesis of lipoxygenase (LOX) metabolites. Quantitative oxylipin analysis identified less than twofold increased jasmonate levels in healthy fou2 leaves compared to wild-type; however, wounded fou2 leaves strongly increased jasmonate biogenesis compared to wounded wild-type. Furthermore, the plants displayed enhanced resistance to the fungus Botrytis cinerea. Higher than wild-type LOX activity and enhanced resistance in the fou2 mutant depend fully on a functional jasmonate response pathway. The fou2 mutant carries a missense mutation in the putative voltage sensor of the Two Pore Channel 1 gene (TPC1), which encodes a Ca(2+)-permeant non-selective cation channel. Patch-clamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. The results indicate that cation fluxes exert strong control over the positive feedback loop whereby JA stimulates its own synthesis.

Personalized drug administrations using Support Vector Machine

The decision-making process regarding drug dose, regularly used in everyday medical practice, is critical to patients' health and recovery. It is a challenging process, especially for a drug with narrow therapeutic ranges, in which a medical doctor decides the quantity (dose amount) and frequency (dose interval) on the basis of a set of available patient features and doctor's clinical experience (a priori adaptation). Computer support in drug dose administration makes the prescription procedure faster, more accurate, objective, and less expensive, with a tendency to reduce the number of invasive procedures. This paper presents an advanced integrated Drug Administration Decision Support System (DADSS) to help clinicians/patients with the dose computing. Based on a support vector machine (SVM) algorithm, enhanced with the random sample consensus technique, this system is able to predict the drug concentration values and computes the ideal dose amount and dose interval for a new patient. With an extension to combine the SVM method and the explicit analytical model, the advanced integrated DADSS system is able to compute drug concentration-to-time curves for a patient under different conditions. A feedback loop is enabled to update the curve with a new measured concentration value to make it more personalized (a posteriori adaptation).

How a haemosporidian parasite of bats gets around: the genetic structure of a parasite, vector and host compared.

Parasite population structure is often thought to be largely shaped by that of its host. In the case of a parasite with a complex life cycle, two host species, each with their own patterns of demography and migration, spread the parasite. However, the population structure of the parasite is predicted to resemble only that of the most vagile host species. In this study, we tested this prediction in the context of a vector-transmitted parasite. We sampled the haemosporidian parasite Polychromophilus melanipherus across its European range, together with its bat fly vector Nycteribia schmidlii and its host, the bent-winged bat Miniopterus schreibersii. Based on microsatellite analyses, the wingless vector, and not the bat host, was identified as the least structured population and should therefore be considered the most vagile host. Genetic distance matrices were compared for all three species based on a mitochondrial DNA fragment. Both host and vector populations followed an isolation-by-distance pattern across the Mediterranean, but not the parasite. Mantel tests found no correlation between the parasite and either the host or vector populations. We therefore found no support for our hypothesis; the parasite population structure matched neither vector nor host. Instead, we propose a model where the parasite's gene flow is represented by the added effects of host and vector dispersal patterns.

Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma.

BACKGROUND: HOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma. RESULTS: We observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively. CONCLUSIONS: Based on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

Interleukin-1- and type I interferon-dependent enhanced immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletions of type I and type II interferon-binding proteins.

UNLABELLED: NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4(+) T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV vaccine vector. IMPORTANCE: NYVAC is a replication-deficient poxvirus developed as a vaccine vector against HIV. NYVAC expresses several genes known to impair the host immune defenses by interfering with innate immune receptors, cytokines, or interferons. Given the crucial role played by interferons against viruses, we postulated that targeting the type I and type II decoy receptors used by poxvirus to subvert the host innate immune response would be an attractive approach to improve the immunogenicity of NYVAC vectors. Using systems biology approaches, we report that deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus resulted in the robust expression of type I IFNs and interferon-stimulated genes (ISGs), a strong activation of the inflammasome, and upregulated expression of IL-1β and proinflammatory cytokines. Dual deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus improves its immunogenic profile and makes it an attractive candidate HIV vaccine vector.

What Role Does Sleep Play in Weight Gain in the First Semester of University?

We hypothesized that shorter sleep durations and greater variability in sleep patterns are associated with weight gain in the first semester of university. Students (N = 132) completed daily sleep diaries for 9 weeks, completed the MEQ (chronotype) and CES-D (depressed mood) at week 9, and self-reported weight/height (weeks 1 & 9). Mean and variability scores were calculated for sleep duration (TST, TSTv), bedtime (BT, BTv), and wake time (WT, WTv). An initial hierarchical regression evaluated (block 1) sex, ethnicity; (block 2) depressed mood, chronotype; (block 3) TST; (block 4) BT, WT; and (block 5; R(2) change = 0.09, p = 0.005) TSTv, BTv, WTv with weight change. A sex-by-TSTv interaction was found. A final model showed that ethnicity, TST, TSTv, and BTv accounted for 31% of the variance in weight change for males; TSTv was the most significant contributor (R(2) change = 0.21, p < 0.001). Daily variability in sleep duration contributes to males' weight gain. Further investigation needs to examine sex-specific outcomes for sleep and weight.

Signs of a vector's adaptive choice: on the evasion of infectious hosts and parasite-induced mortality

Laboratory and field experiments have demonstrated in many cases that malaria vectors do not feed randomly, but show important preferences either for infected or non-infected hosts. These preferences are likely in part shaped by the costs imposed by the parasites on both their vertebrate and dipteran hosts. However, the effect of changes in vector behaviour on actual parasite transmission remains a debated issue. We used the natural associations between a malaria-like parasite Polychromophilus murinus, the bat fly Nycteribia kolenatii and a vertebrate host the Daubenton's bat Myotis daubentonii to test the vector's feeding preference based on the host's infection status using two different approaches: 1) controlled behavioural assays in the laboratory where bat flies could choose between a pair of hosts; 2) natural bat fly abundance data from wild-caught bats, serving as an approximation of realised feeding preference of the bat flies. Hosts with the fewest infectious stages of the parasite were most attractive to the bat flies that did switch in the behavioural assay. In line with the hypothesis of costs imposed by parasites on their vectors, bat flies carrying parasites had higher mortality. However, in wild populations, bat flies were found feeding more based on the bat's body condition, rather than its infection level. Though the absolute frequency of host switches performed by the bat flies during the assays was low, in the context of potential parasite transmission they were extremely high. The decreased survival of infected bat flies suggests that the preference for less infected hosts is an adaptive trait. Nonetheless, other ecological processes ultimately determine the vector's biting rate and thus transmission. Inherent vector preferences therefore play only a marginal role in parasite transmission in the field. The ecological processes rather than preferences per se need to be identified for successful epidemiological predictions.

Importance of early weight changes to predict long-term weight gain during psychotropic drug treatment.

BACKGROUND: Psychotropic drugs can induce substantial weight gain, particularly during the first 6 months of treatment. The authors aimed to determine the potential predictive power of an early weight gain after the introduction of weight gain-inducing psychotropic drugs on long-term weight gain. METHOD: Data were obtained from a 1-year longitudinal study ongoing since 2007 including 351 psychiatric (ICD-10) patients, with metabolic parameters monitored (baseline and/or 1, 3, 6, 9, 12 months) and with compliance ascertained. International Diabetes Federation and World Health Organization definitions were used to define metabolic syndrome and obesity, respectively. RESULTS: Prevalences of metabolic syndrome and obesity were 22% and 17%, respectively, at baseline and 32% and 24% after 1 year. Receiver operating characteristic analyses indicated that an early weight gain > 5% after a period of 1 month is the best predictor for important long-term weight gain (≥ 15% after 3 months: sensitivity, 67%; specificity, 88%; ≥ 20% after 12 months: sensitivity, 47%; specificity, 89%). This analysis identified most patients (97% for 3 months, 93% for 12 months) who had weight gain ≤ 5% after 1 month as continuing to have a moderate weight gain after 3 and 12 months. Its predictive power was confirmed by fitting a longitudinal multivariate model (difference between groups in 1 year of 6.4% weight increase as compared to baseline, P = .0001). CONCLUSION: Following prescription of weight gain-inducing psychotropic drugs, a 5% threshold for weight gain after 1 month should raise clinician concerns about weight-controlling strategies.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.