Interaction of synthetic antimicrobial peptides of the Hylin a1 family with models of eukaryotic structures: Zwitterionic membranes and DNA

Antimicrobial peptides (AMPs) have been appointed as a possible alternative to traditional antibiotics in face of pathogens increasing resistance to conventional drugs. Hylin a1 (IFGAILPLALGALKNLIK), an AMP extracted from the skin secretion of a South American frog, Hypsiboas albopunctatus, was found to show a strong cytotoxicity against bacteria and fungus, but also a considerable hemolytic action. Considering the toxicity of the peptide in eukaryotic cells, this work focuses on investigating the effects of the interaction of the Hylin a1 analogues W⁶Hya1, D⁰W⁶Hya1 and K⁰W⁶Hya1 with models of eukaryotic structures, namely zwitterionic liposomes of dipalmitoyl phosphatidylcholine (DPPC) and calf-thymus DNA (CT DNA). Through intrinsic Trp fluorescence we determined that the peptide affinity for fluid DPPC bilayers follows the decreasing order: D⁰W⁶Hya1 (+2) > W⁶Hya1 (+3) » K⁰W⁶Hya1 (+4). Fluorescence data also indicate that the Trp residue in the more positively charged peptide, K⁰W⁶Hya1, is less deep in the bilayer than the residue in the other two peptides. This finding is supported by differential scanning calorimetry (DSC) data, which shows that both D⁰W⁶Hya1 and W⁶Hya1 disturb DPPC gel-fluid transition slightly more effectively than K⁰W⁶Hya1. DPPC DSC profiles are homogeneously disturbed by the three peptides, probably related to peptide-membrane diffusion. Surprisingly, the peptide that displays the lowest affinity for PC membranes and is located at the more superficial position in the bilayer, K⁰W⁶Hya1, is the most efficient in causing formation of pores on the membrane, as attested by carboxyfluorescein leakage assays. The three peptides were found to interact with CT DNA, with a deep penetration of the Trp residue into hydrophobic pockets of the double helix, as indicated by the significant blue shift on the Trp fluorescence, and the displacement of DNA-bound ethidium bromide by the peptides. The experiments of DNA electrophoresis confirm that Hylin peptides bind DNA in a concentration-dependent manner, inducing complete DNA retardation at the relative AMP/plasmid DNA weight ratio of ~17. These findings could help to better understand the AMPs toxic effects on eukaryotic cells, thus contributing to the design of healthier therapeutic agents.     

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

Background

Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background.

Results

Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type.

Conclusions

The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.

     

X-linked adrenoleukodystrophy (X-ALD): clinical presentation and guidelines for diagnosis, follow-up and management

Background

Pompe disease (Glycogen storage disease type II, GSD II, acid alpha-glucosidase deficiency, acid maltase deficiency, OMIM # 232300) is an autosomal-recessive lysosomal storage disorder due to a deficiency of acid alpha-glucosidase (GAA, acid maltase, EC 3.2.1.20, Swiss-Prot P10253). Clinical manifestations are dominated by progressive weakness of skeletal muscle throughout the clinical spectrum. In addition, the classic infantile form is characterised by hypertrophic cardiomyopathy.

Methods

In a cross-sectional single-centre study we clinically assessed 3 patients with classic infantile Pompe disease and 39 patients with non-classic presentations, measured their acid alpha-glucosidase activities and analysed their GAA genes.

Results

Classic infantile patients had nearly absent residual enzyme activities and a typical clinical course with hypertrophic cardiomyopathy until the beginning of therapy. The disease manifestations in non-classic patients were heterogeneous. There was a broad variability in the decline of locomotive and respiratory function. The age of onset ranged from birth to late adulthood and correlated with enzyme activities. Molecular analysis revealed as many as 33 different mutations, 14 of which are novel. All classic infantile patients had two severe mutations. The most common mutation in the non-classic group was c.-32-13?T?>?G. It was associated with a milder course in this subgroup.

Conclusions

Disease manifestation strongly correlates with the nature of the GAA mutations, while the variable progression in non-classic Pompe disease is likely to be explained by yet unknown modifying factors. This study provides the first comprehensive dataset on the clinical course and the mutational spectrum of Pompe disease in Germany.     

Effect of Providing Multiple Micronutrients in Powder through Primary Healthcare on Anemia in Young Brazilian Children: A Multicentre Pragmatic Controlled Trial

Background

Multiple micronutrients in powder (MNP) are recommended by WHO to prevent anemia in young children. However, evidences for its effectiveness in different populations and improvements in other outcomes (e.g. linear growth and vitamin A deficiency) are scarce.

Methods

A multicentre pragmatic controlled trial was carried out in primary health centres. At study baseline, a control group (CG) of children aged 10- to 14 months (n = 521) was recruited in the routine healthcare for assessing anemia, anthropometric and micronutrient status. At the same time, an intervention group (IG) of infants aged 6- to 8 months (n = 462) was recruited to receive MNP daily in complementary feeding over a period of 60 days. Both study groups were compared when the IG infants reached the age of the CG children at enrolment.

Results

In CG, the prevalence of anemia [hemoglobin (Hb) < 110 g/L], iron deficiency (ID, plasma ferritin < 12 μg/L or TfR > 8.3 mg/L), and vitamin A deficiency (VAD, serum retinol < 0.70μmol/L) were 23.1%, 37.4%, and 17.4%, respectively. Four to six months after enrolment, when the IG participants had the same age of the controls at the time of testing, the prevalence of anemia, ID and VAD in IG were 14.3%, 30.1% and 7.9%, respectively. Adjusting for city, health centre, maternal education, and age, IG children had a lower likelihood of anemia and VAD [Prevalence Ratio (95% CI) = 0.63 (0.45, 0.88) and 0.45 (0.29, 0.69), respectively] when compared with CG children. The adjusted mean distributions of Hb and length-for-age Z-scores improved by 2 SE in the IG compared to CG children.

Conclusions

MNP effectively reduced anemia and improved growth and micronutrient status among young Brazilian children.

Trial Registration

Registro Brasileiro de Ensaios Clinicos RBR-5ktv6b     

A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA

Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.     

In vitro test systems supporting the development of improved pest control methods: a case study with chemical mixtures and bivalve biofoulers

Using the bivalve macrofouler Corbicula fluminea, the suitability of in vitro testing as a stepping stone towards the improvement of control methods based on chemical mixtures was addressed in this study. In vitro cholinesterase (ChE) activity inhibition following single exposure of C. fluminea tissue to four model chemicals (the organophosphates dimethoate and dichlorvos, copper and sodium dodecyl phosphate [SDS]) was first assessed. Consequently, mixtures of dimethoate with copper and dichlorvos with SDS were tested and modelled; mixtures with ChE revealed synergistic interactions for both chemical pairs. These synergic combinations were subsequently validated in vivo and the increased control potential of these selected combinations was verified, with gains of up to 50% in C. fluminea mortality relative to corresponding single chemical treatments. Such consistency supports the suitability of using time- and cost-effective surrogate testing platforms to assist the development of biofouling control strategies incorporating mixtures.     

How do leaf wetting events affect gas exchange and leaf lifespan of plants from seasonally dry tropical vegetation?

• Foliar uptake of dew is likely an important mechanism of water acquisition for plants from tropical dry environments. However, there is still limited experimental evidence describing the anatomical pathways involved in this process and the effects of this water subsidy on the maintenance of gas exchange and leaf lifespan of species from seasonally dry tropical vegetation such as the Brazilian caatinga.
• We performed scanning electron, bright‐field and confocal microscopic analyses and used apoplastic tracers to examine the foliar water uptake (FWU) routes in four woody species with different foliar phenology and widely distributed in the caatinga. Leaves of plants subjected to water stress were exposed to dew simulation to evaluate the effects of the FWU on leaf water potentials, gas exchange and leaf lifespan.
• All species absorbed water through their leaf cuticles and/or peltate trichomes but FWU capacity differed among species. Leaf wetting by dew increased leaf lifespan duration up to 36 days compared to plants in the drought treatment. A positive effect on leaf gas exchange and new leaf production was only observed in the anisohydric and evergreen species.
• We showed that leaf wetting by dew is relevant for the physiology and leaf lifespan of plants from seasonally dry tropical vegetation, especially for evergreen species.

     

Isolation and characterization of dinucleotide microsatellite loci in communally breeding Guira cuckoos (Aves: Cuculidae)

Guira cuckoos (Guira guira) are communal nesting birds endemic to South America that show high levels of conflict between members of the group over the contribution to the clutch. Adults eject eggs and even nestlings from the communal nest, sometimes leading to the loss of the entire brood. We developed seven polymorphic microsatellites for Guira cuckoos using an enrichment protocol. The number of alleles ranged from 5 to 14 (mean 9.86) and the heterozygosity ranged from 0.41 to 0.89 for the eight to 88 individuals screened. These loci will allow parentage assignments and population analysis in this species.