Distilling a Visual Network of Retinitis Pigmentosa Gene-Protein Interactions to Uncover New Disease Candidates

Background

Retinitis pigmentosa (RP) is a highly heterogeneous genetic visual disorder with more than 70 known causative genes, some of them shared with other non-syndromic retinal dystrophies (e.g. Leber congenital amaurosis, LCA). The identification of RP genes has increased steadily during the last decade, and the 30% of the cases that still remain unassigned will soon decrease after the advent of exome/genome sequencing. A considerable amount of genetic and functional data on single RD genes and mutations has been gathered, but a comprehensive view of the RP genes and their interacting partners is still very fragmentary. This is the main gap that needs to be filled in order to understand how mutations relate to progressive blinding disorders and devise effective therapies.

Methodology

We have built an RP-specific network (RPGeNet) by merging data from different sources: high-throughput data from BioGRID and STRING databases, manually curated data for interactions retrieved from iHOP, as well as interactions filtered out by syntactical parsing from up-to-date abstracts and full-text papers related to the RP research field. The paths emerging when known RP genes were used as baits over the whole interactome have been analysed, and the minimal number of connections among the RP genes and their close neighbors were distilled in order to simplify the search space.

Conclusions

In contrast to the analysis of single isolated genes, finding the networks linking disease genes renders powerful etiopathological insights. We here provide an interactive interface, RPGeNet, for the molecular biologist to explore the network centered on the non-syndromic and syndromic RP and LCA causative genes. By integrating tissue-specific expression levels and phenotypic data on top of that network, a more comprehensive biological view will highlight key molecular players of retinal degeneration and unveil new RP disease candidates.     

In Vitro Activity of Rifampicin and Verapamil Combination in Multidrug-Resistant Mycobacterium tuberculosis

The aim of the present study was to evaluate the effect of the combination of rifampicin (RIF) and verapamil (VP) against the Mycobacterium tuberculosis H₃₇Rv reference strain and six multidrug-resistant (MDR) M. tuberculosis clinical isolates by determining Time-Kill Curves and the ability to efflux drug by fluorometry. The RIF+VP combination showed synergism in one MDR clinical isolate. For the other five MDR clinical isolates, the drug combination showed no interaction. The MDR clinical isolate had lower ethidium bromide (EtBr) accumulation when exposed to the RIF+VP combination, compared with RIF and VP exposure alone. The other MDR clinical isolates showed no significant difference in EtBr accumulation. These results suggest greater efflux action in one of the MDR clinical isolates compared with the M. tuberculosis H₃₇Rv reference strain. The other five MDR isolates may have additional mechanisms of drug resistance to RIF. The use of the RIF+VP combination made one MDR bacillus more susceptible to RIF probably by inhibiting efflux pumps, and this combination therapy, in some cases, may contribute to a reduction of resistance to RIF in M. tuberculosis.     

Management of a caseous lymphadenitis outbreak in a new Iberian ibex (<Emphasis Type="Italic">Capra pyrenaica</Emphasis>) stock reservoir

Background

In 2010, an Iberian ibex (Capra pyrenaica hispanica) stock reservoir was established for conservation purposes in north-eastern Spain. Eighteen ibexes were captured in the wild and housed in a 17 hectare enclosure. Once in captivity, a caseous lymphadenitis (CLA) outbreak occurred and ibex handlings were carried out at six-month intervals between 2010 and 2013 to perform health examinations and sampling. Treatment with a bacterin-based autovaccine and penicillin G benzatine was added during the third and subsequent handlings, when infection by Corynebacterium pseudotuberculosis was confirmed. Changes in lesion score, serum anti-C. pseudotuberculosis antibodies and haematological parameters were analyzed to assess captivity effects, disease emergence and treatment efficacy. Serum acute phase proteins (APP) Haptoglobin (Hp), Amyloid A (SAA) and Acid Soluble Glycoprotein (ASG) concentrations were also determined to evaluate their usefulness as indicators of clinical status.Once in captivity, 12 out of 14 ibexes (85.7%) seroconverted, preceding the emergence of clinical signs; moreover, TP, WBC, eosinophil and platelet cell counts increased while monocyte and basophil cell counts decreased. After treatment, casualties and fistulas disappeared and both packed cell volume (PCV) and haemoglobin concentration significantly increased. Hp, SAA and ASG values were under the limit of detection or showed no significant differences.

Conclusions

A role for captivity in contagion rate is suggested by the increase in antibody levels against C. pseudotuberculosis and the emergence of clinical signs. Although boosted by captivity, this is the first report of an outbreak of caseous lymphadenitis displaying high morbidity and mortality in wild ungulates. Treatment consisting of both vaccination and antibiotic therapy seemed to prevent mortality and alleviate disease severity, but was not reflected in the humoural response. Haematology and APP were not useful indicators in our study, perhaps due to the sampling frequency. Presumably endemic and irrelevant in the wild, this common disease of domestic small ruminants is complicating conservation efforts for the Iberian ibex in north-eastern Spain.

     

Effect of Maraviroc Intensification on HIV-1-Specific T Cell Immunity in Recently HIV-1-Infected Individuals

Background

The effect of maraviroc on the maintenance and the function of HIV-1-specific T cell responses remains unknown.

Methods

Subjects recently infected with HIV-1 were randomized to receive anti-retroviral treatment with or without maraviroc intensification for 48 weeks, and were monitored up to week 60. PBMC and in vitro-expanded T cells were tested for responses to the entire HIV proteome by ELISpot analyses. Intracellular cytokine staining assays were conducted to monitor the (poly)-functionality of HIV-1-specific T cells. Analyses were performed at baseline and week 24 after treatment start, and at week 60 (3 months after maraviroc discontinuation).

Results

Maraviroc intensification was associated with a slower decay of virus-specific T cell responses over time compared to the non-intensified regimen in both direct ex-vivo as well as in in-vitro expanded cells. The effector function profiles of virus-specific CD8⁺ T cells were indistinguishable between the two arms and did not change over time between the groups.

Conclusions

Maraviroc did not negatively impact any of the measured parameters, but was rather associated with a prolonged maintenance of HIV-1-specific T cell responses. Maraviroc, in addition to its original effect as viral entry inhibitor, may provide an additional benefit on the maintenance of virus-specific T cells which may be especially important for future viral eradication strategies.     

Detection and quantification of farnesol-induced apoptosis in difficult primary cell cultures by TaqMan protein assay

Apoptosis can be detected reliably by assaying for cleaved caspase-3, for which active caspase-3 antibodies are used in several methods, such as immunocytochemistry, enzyme-linked immunosorbent assay, and western blot. In this study, we used TaqMan protein assay (TPA), a novel method for protein detection and quantification that detects proteins by amplification of substitute DNA templates. TPA uses antibodies and proximity ligation for quantitative real-time PCR. Meningiomas are primarily benign intracranial tumors. Primary cell cultures of meningiomas are often unsuitable for sensitive protein detection methods. We optimized a TPA to detect active caspase-3 and evaluated its ability to detect farnesol-induced apoptosis in primary meningioma cells. The specificity and sensitivity of the inactive and active caspase-3 assay were determined using recombinant caspase-3. Apoptosis was induced in meningiomas in the presence of 0.2 μM farnesol as shown by immunocytochemistry of single-stranded DNA. Also, viability decreased by over 90 % after treatment with 1.2 μM farnesol for 24 h. The TPA detected a significant increase in active caspase-3 after treatment with 2 and 4 μM farnesol for 2 h, which could not be detected using standard methods such as western blot and immunofluorescence. In addition, TPA determined that meningiomas show disparate sensitivities to low concentrations of farnesol. Caspase-3 expression fell significantly in cells that were treated with 0.25 μM farnesol for 2 h. Further, by TPA, active caspase-3 peaked after 2 h and declined with longer incubation times. This study demonstrates that cleaved caspase-3 is detected and quantified reliably in meningiomas by TPA.     

Complex formation of chlorhexidine gluconate with hydroxypropyl-β-cyclodextrin (HPβCD) by proton nuclear magnetic resonance spectroscopy ((1)H NMR)

The complex formation of chlorhexidine digluconate (CHX-G(2)) with hydroxypropyl-β-cyclodextrin (HPβCD) was studied using NMR spectroscopy. The results revealed that this surfactant agent shows an monomer/aggregate equilibrium, which is dependent on the concentration of this drug. This equilibrium can be modified by the presence of HPβCD, which reduces the aggregation of the CHX-G(2) molecules. An inclusion process of the CHX-G(2) aromatic residue within the cyclodextrin cavity was confirmed by 2D ROESY spectroscopy. (1)H NMR titration studies of CHX-G(2) with HPβCD in D(2)O confirmed the formation of higher order complexes between CHX-G(2) and HPβCD. Moreover, the addition of HPβCD into CHX-G(2) solutions forms insoluble aggregates. Such insoluble aggregates may result in the stacking of CHX-G(2) molecules on the surface of the CHX-G(2):HPβCD complexes.     

Complex formation of chlorhexidine gluconate with hydroxypropyl-β-cyclodextrin (HPβCD) by proton nuclear magnetic resonance spectroscopy (H NMR)

The complex formation of chlorhexidine digluconate (CHX-G₂) with hydroxypropyl-β-cyclodextrin (HPβCD) was studied using NMR spectroscopy. The results revealed that this surfactant agent shows an monomer/aggregate equilibrium, which is dependent on the concentration of this drug. This equilibrium can be modified by the presence of HPβCD, which reduces the aggregation of the CHX-G₂ molecules. An inclusion process of the CHX-G₂ aromatic residue within the cyclodextrin cavity was confirmed by 2D ROESY spectroscopy. ¹H NMR titration studies of CHX-G₂ with HPβCD in D₂O confirmed the formation of higher order complexes between CHX-G₂ and HPβCD. Moreover, the addition of HPβCD into CHX-G₂ solutions forms insoluble aggregates. Such insoluble aggregates may result in the stacking of CHX-G₂ molecules on the surface of the CHX-G₂:HPβCD complexes.     

Neuropeptide‐S‐receptor deficiency affects sex‐specific modulation of safety learning by pre‐exposure to electric stimuli

Neuropeptide S (NPS) is a neuropeptide involved in the regulation of fear. Because safety learning is impaired in patients suffering from anxiety‐related psychiatric disorders, and polymorphisms of the human neuropeptide S receptor (NPSR) gene have also been associated with anxiety disorders, we wanted to investigate whether NPSR‐deficiency interferes with safety learning, and how prior stress would affect this type of learning. We first investigated the effect of pre‐exposure to two different types of stressors (electric stimuli or immobilization) on safety learning in female and male C57Bl/6 mice, and found that while stress induced by electric stimuli enhanced safety learning in males, there were no differences in safety learning following immobilization stress. To further investigate the role of the NPS system in stress‐induced modulation of safety learning, we exposed NPSR‐deficient mice to stress induced by electric stimuli 10 days before safety learning. In nonstressed male mice, NPSR‐deficiency enhanced safety learning. As in male C57Bl/6 mice, pre‐exposure to electric stimuli increased safety learning in male NPSR +/+ mice. This pre‐exposure effect was blocked in NPSR‐deficient male mice showing impaired, but still intact, safety learning in comparison to their NPSR +/+ and NPSR +/? littermates. There was neither a pre‐exposure nor a genotype effect in female mice. Our findings provide evidence that pre‐exposure to stress induced by electric stimuli enhances safety learning in male mice, and that NPSR‐deficiency prevents the beneficial effect of stress exposure on safety learning. We propose an inverted U‐shape relationship between stress and safety learning.