Contributions of different mosquito species to the transmission of lymphatic filariasis in central Nigeria: implications for monitoring infection by PCR in mosquito pools

Background

Monitoring and evaluation are essential to the successful implementation of mass drug administration programmes for LF elimination. Monitoring transmission when it is low requires both large numbers of mosquito vectors and sensitive methods for detecting Wuchereria bancrofti infections in them. PCR-based methods are preferred over classical dissections but the best protocol so far achieved detection of one L₃ Wuchereria bancrofti larva in a pool of 35–50 Anopheles mosquitoes. It also lacks consistency and remains still a costly tool. Hence we decided to improve upon this to achieve detection in a pool of 100 or more by enhancing the quality of the template DNA. Prior to this we also evaluated three vector sampling methods in the context of numbers for monitoring.

Methods

Human landing, pyrethrium spray and light traps catches were conducted concurrently at sites in an LF endemic district in Ghana and the numbers obtained compared. Two DNA extraction methods; Bender buffer and phenol/chloroform purification, and DNAeasy Tissue kit (Quaigen Inc) were used on pools of 25, 50, 75 100 and 150 mosquitoes each seeded with one L₃ or its quivalent amount of DNA. Then another set of extracted DNA by the two methods was subjected to Dynal bead purification method (using capture oligonucleotide primers). These were used as template DNA in PCR to amplify W. bancrofti sequences. The best PCR result was then evaluated in the field at five sites by comparing its results (infections per 1000 mosquitoes) with that of dissection of roughly equal samples sizes.

Results

The largest numbers of mosquitoes were obtained with the human landing catches at all the sites sampled. Although PCR detection of one L₃ in pools of 25, 50 and 75 mosquitoes was consistent irrespective of the extraction method, that of one L₃ in 100 was only achieved with the kit-extracted DNA/Dynal bead purification method. Infections were found at only two sites by both dissection and pool-screening being 14.3 and 19 versus 13.4 and 20.1 per 1000 Anopheles mosquitoes respectively, which were not statistically significant

Discussion and conclusion

HLC still remains the best option for sampling for the large numbers of mosquitoes required for monitoring transmission during MDA programmes, when vector population densities are high and classical indices of transmission are required. One – in – 100 detection is an improvement on previous PCR pool-screening methods, which in our opinion was a result of the introduction of the extra step of parasite DNA capture using Dynal/beads. As pool sizes increase the insects DNA will swamp parasite DNA making the latter less available for an efficient PCR, therefore we propose either additional steps of parasite DNA capture or real-time PCR to improve further the pool screening method. The study also attests also to the applicability of Katholi et al's algorithm developed for determining onchocerciasis prevalence in LF studies.     

A rapid microassay to evaluate enzymatic hydrolysis of lignocellulosic substrates

Current attempts to produce ethanol from lignocellulosic biomass are focused on the optimization of pretreatment to reduce substrate recalcitrance and the improvement of enzymes for hydrolysis of the cellulose and hemicellulose components to produce fermentable sugars. Research aimed at optimizing both aspects of the bioconversion process involves assessment of the effects of multiple variables on enzyme efficiency, resulting in large factorial experiments with intensive assay requirements. A rapid assay for lignocellulose hydrolysis has been developed to address this need. Pretreated lignocellulose is formed into handsheets, which are then used to prepare small disks that are easily dispensed into microtiter plates. The hydrolysis of cellulose to glucose is estimated using an enzyme-coupled spectrophotometric assay. Using disks prepared from ethanol organosolv pretreated yellow poplar, it is shown that the assay generates data comparable with those produced by hydrolysis of pretreated yellow poplar pulp in Erlenmeyer flasks, followed by HPLC analysis of glucose. The assay shows considerable time and cost benefits over the standard assay protocol and is applicable to a broad range of lignocellulosic substrates.     

Novel histamine H3 receptor antagonists based on the 4-[(1H-imidazol-4-yl)methyl]piperidine scaffold

We report the discovery of novel histamine H(3) receptor antagonists based on 4-[(1H-imidazol-4-yl)methyl]piperidine. The most potent compounds in the series (e.g., 7) result from the attachment of a substituted aniline amide to the main pharmacophore piperidine via a two-methylene linker.     

The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.     

Notch ligand delta-like 4 regulates development and pathogenesis of allergic airway responses by modulating IL-2 production and Th2 immunity

Activation of the canonical Notch pathways has been implicated in Th cell differentiation, but the role of specific Notch ligands in Th2-mediated allergic airway responses has not been completely elucidated. In this study, we show that delta-like ligand 4 (Dll4) was upregulated on dendritic cells in response to cockroach allergen. Blocking Dll4 in vivo during either the primary or secondary response enhanced allergen-induced pathogenic consequences including airway hyperresponsiveness and mucus production via increased Th2 cytokines. In vitro assays demonstrated that Dll4 regulates IL-2 in T cells from established Th2 responses as well as during primary stimulation. Notably, Dll4 blockade during the primary, but not the secondary, response increased IL-2 levels in lung and lymph node of allergic mice. The in vivo neutralization of Dll4 was associated with increased expansion and decreased apoptosis during the primary allergen sensitization. Moreover, Dll4-mediated Notch activation of T cells during primary stimulation in vitro increased apoptosis during the contraction/resting phase of the response, which could be rescued by exogenous IL-2. Consistent with the role for Dll4-mediated IL-2 regulation in overall T cell function, the frequency of IL-4-producing cells was also significantly altered by Dll4 both in vivo and in vitro. These data demonstrate a regulatory role of Dll4 both in initial Th2 differentiation and in Th2 cytokine production in established allergic responses.