Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes

The optimal ratio of the polycation's amine to DNA phosphate group (N:P) for efficient polymer-based transfection always employs excess polycation versus DNA. Most of the excess polycation remains free in solution, unassociated with the polyplexes, but is essential for efficient transfection. The mechanism by which excess polycation increases transfection efficiency is not identified. We hypothesised that excess chitosan facilitates intracellular lysosomal escape of the polyplexes. We highlight here the essential role of excess chitosan by rescuing poorly transfecting low N:P ratio polyplexes, by adding free chitosan before or after polyplex addition to cells. We examined polyplex uptake, the kinetics of rescue, intracellular trafficking, and the effects of lysosomotropic agents. We found the facilitating role of excess chitosan to be downstream of cellular uptake. Live-cell confocal quantification of intracellular trafficking revealed prolonged colocalisation of low N:P polyplexes within lysosomes, compared to shorter residence times for both rescued or N:P 5 samples, followed by observation of free pDNA in the cytosol. These data demonstrate that excess polycation mediates enhanced transfection efficiency by promoting the release of polyplexes from the endo-lysosomal vesicles, revealing a critical intracellular barrier overcome by excess polycation and suggesting possible avenues for further optimisation of polymer-based gene delivery.     

Genetic architecture of circulating lipid levels

Serum concentrations of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs) and total cholesterol (TC) are important heritable risk factors for cardiovascular disease. Although genome-wide association studies (GWASs) of circulating lipid levels have identified numerous loci, a substantial portion of the heritability of these traits remains unexplained. Evidence of unexplained genetic variance can be detected by combining multiple independent markers into additive genetic risk scores. Such polygenic scores, constructed using results from the ENGAGE Consortium GWAS on serum lipids, were applied to predict lipid levels in an independent population-based study, the Rotterdam Study-II (RS-II). We additionally tested for evidence of a shared genetic basis for different lipid phenotypes. Finally, the polygenic score approach was used to identify an alternative genome-wide significance threshold before pathway analysis and those results were compared with those based on the classical genome-wide significance threshold. Our study provides evidence suggesting that many loci influencing circulating lipid levels remain undiscovered. Cross-prediction models suggested a small overlap between the polygenic backgrounds involved in determining LDL-C, HDL-C and TG levels. Pathway analysis utilizing the best polygenic score for TC uncovered extra information compared with using only genome-wide significant loci. These results suggest that the genetic architecture of circulating lipids involves a number of undiscovered variants with very small effects, and that increasing GWAS sample sizes will enable the identification of novel variants that regulate lipid levels.     

The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein

The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs.     

In vitro antiplasmodial activity and cytotoxicity of crude extracts and compounds from the stem bark of <Emphasis Type="Italic">Kigelia africana</Emphasis> (Lam.) Benth (Bignoniaceae)

In order to assess the potential of the stem bark of Kigelia africana (Lam.) Benth as source of new anti-malarial leads, n-hexane and ethyl acetate (EtOAc) extracts and four compounds isolated from the stem bark were screened in vitro against the chloroquine-resistant W-2 and two field isolates of Plasmodium falciparum using lactate dehydrogenase assay. The products were also tested for their cytotoxicity on LLC/MK2 monkey kidney cells. The EtOAc extract exhibited a significant antiplasmodial activity (IC₅₀ = 11.15 μg/mL on W-2; 3.91 and 4.74 μg/mL on field CAM10 and SHF4 isolates, respectively), whereas the n-hexane fraction showed a weak activity (IC₅₀ = 73.78 μg/mL on W-2 and 21.85 μg/mL on SHF4). Three out of the four compounds showed good activity against all the three different parasite strains (IC₅₀ < 5 μM). Specicoside exhibited the highest activity on W-2 (IC₅₀ = 1.54 μM) followed by 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid (IC₅₀ = 1.60 μM) and atranorin (IC₅₀ = 4.41 μM), while p-hydroxycinnamic acid was the least active (IC₅₀ = 53.84 μM). The EtOAc extract and its isolated compounds (specicoside and p-hydroxycinnamic acid) were non-cytotoxic (CC₅₀ > 30 μg/mL), whereas the n-hexane extract and two of its products, atranorin and 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid showed cytotoxicity at high concentrations, with the last one being the most toxic (CC₅₀ = 9.37 μg/mL). These findings justify the use of K. africana stem bark as antimalaria by traditional healers of Western Cameroon, and could constitute a good basis for further studies towards development of new leads or natural drugs for malaria.     

HTLV infection among foreign pregnant women living in Spain

Background

The overall seroprevalence of HTLV infection among pregnant women in Spain is below 0.02% and accordingly universal antenatal screening is not recommended. However, as the number of immigrants has significantly increased during the last decade, this population might warrant specific considerations.

Objective

To evaluate the seroprevalence of HTLV infection among immigrant pregnant women living in Spain.

Methods

From January 2009 to December 2010 a cross-sectional study was carried out in all foreign pregnant women attended at 14 Spanish clinics. All were tested for HTLV antibodies using a commercial enzyme-immunoassay, being reactive samples confirmed by Western blot or PCR.

Results

A total of 3337 foreign pregnant women were examined. Their origin was as follows: Latin America 1579 (47%), North Africa 507 (16%), East Europe 606 (18%), Sub-Saharan Africa 316 (9%), North America and West Europe 116 (3.5%) and Asia and Australia 163 (5%). A total of 7 samples were confirmed as HTLV positive, of which 6 were HTLV-1 and 1 HTLV-2. HTLV-1 infection was found in 5 women coming from Latin America and 1 from Morocco. The only woman with HTLV-2 came from Ghana. The overall HTLV seroprevalence was 0.2%, being 0.3% among Latin Americans and 0.2% among Africans. It was absent among women coming from other regions.

Conclusions

The seroprevalence of HTLV infection among foreign pregnant women in Spain is 0.2%, being all cases found in immigrants from Latin America and Africa. Given the benefit of preventing vertical transmission, antenatal screening should be recommended in pregnant women coming from these regions.     

Developing an electronic health record (EHR) for methadone treatment recording and decision support

Background  

In this paper, we give an overview of methadone treatment in Ireland and outline the rationale for designing an electronic health record (EHR) with extensibility, interoperability and decision support functionality. Incorporating several international standards, a conceptual model applying a problem orientated approach in a hierarchical structure has been proposed for building the EHR.     

Heterozygous COL9A3 variants cause severe peripheral vitreoretinal degeneration and retinal detachment

The COL9A3 gene encodes one of the three alpha chains of Type IX collagen, with heterozygous variants reported to cause multiple epiphyseal dysplasia, and suggested as contributory in some cases of sensorineural hearing loss. Patients with homozygous variants have midface hypoplasia, myopia, sensorineural hearing loss, epiphyseal changes and carry a diagnosis of Stickler syndrome. Variants in COL9A3 have not previously been reported to cause vitreoretinal degeneration and/or retinal detachments. This report describes two families with autosomal dominant inheritance and predominant features of peripheral vitreoretinal lattice degeneration and retinal detachment. Genomic sequencing revealed a heterozygous splice variant in COL9A3 [{"type":"entrez-nucleotide","attrs":{"text":"NG_016353.1","term_id":"283945463"}}NG_016353.1({"type":"entrez-nucleotide","attrs":{"text":"NM_001853.4","term_id":"1432073434"}}NM_001853.4):c.1107 + 1G>C, NC_000020.10({"type":"entrez-nucleotide","attrs":{"text":"NM_001853.4","term_id":"1432073434"}}NM_001853.4):c.1107 + 1G>C, LRG1253t1] in Family 1, and a heterozygous missense variant [{"type":"entrez-nucleotide","attrs":{"text":"NG_016353.1","term_id":"283945463"}}NG_016353.1({"type":"entrez-nucleotide","attrs":{"text":"NM_001853.4","term_id":"1432073434"}}NM_001853.4):c.388G>A p.(Gly130Ser)] in Family 2, each segregating with disease. cDNA studies of the splice variant demonstrated an in-frame deletion in the COL2 domain, and the missense variant occurred in the COL3 domain, both indicating the critical role of Type IX collagen in the vitreous base of the eye.Subject terms: Genetic testing, Medical genetics, Medical genomics     

Skeletal development of Kinosternon scorpioides limbs (Chelonia: Kinosternidae)

The morphological study of limbs is important for the understanding of tetrapod biology, where it can be applied to taxonomy and phylogeny, as well ecology and behavior. In this study area, osteogenesis is a subject in Kinosternidae, which has been little researched. The main aim of this study was to characterize the skeletogenesis of Kinosternon scorpioides limbs. Samples were histologically processed, and the embryos were cleared with potassium hydroxide and stained with alcian blue and alizarin red. It was observed that the limbs arose in embryonic Stage 10 as mesenchymal condensate cells. The first stylopodium chondrification centers were noted at Stage 14. Zeugopodium chondrification centers appeared at Stage 15; carpal, metacarpal, tarsal, and metatarsal regions were observed at Stage 16, and the cartilage molds of all bones limbs were present at Stage 18. Ossification began in the humerus and femur at Stage 20, and continued into the radius, ulna, tibia, and fibula bones. By Stage 23, it was already effectively directed toward the bone epiphyses in both limbs. At Stage 26 and hatching, only articular cartilages remained, and in the majority of samples the carpal region showed no affinity for alcian blue or alizarin red staining. This study acts as an indicative parameter of the taxon's normal development and can contribute to the phylogenetic understanding of this group.     

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis

Background

The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children.

Objective

We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model.

Methods

The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified.

Results

Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis.

Conclusion

We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.