Concentration-dependent, size-independent toxicity of citrate capped AuNPs in Drosophila melanogaster

The expected potential benefits promised by nanotechnology in various fields have led to a rapid increase of the presence of engineered nanomaterials in a high number of commercial goods. This is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of the physical/chemical factors responsible for their toxic effects. In this work, we evaluated the toxicity of monodisperse citrate-capped gold nanoparticles (AuNPs) of different sizes (5, 15, 40, and 80 nm) in the model organism Drosophila melanogaster, upon ingestion. To properly evaluate and distinguish the possible dose- and/or size-dependent toxicity of the AuNPs, we performed a thorough assessment of their biological effects, using two different dose-metrics. In the first approach, we kept constant the total surface area of the differently sized AuNPs (Total Exposed Surface area approach, TES), while, in the second approach, we used the same number concentration of the four different sizes of AuNPs (Total Number of Nanoparticles approach, TNN). We observed a significant AuNPs-induced toxicity in vivo, namely a strong reduction of Drosophila lifespan and fertility performance, presence of DNA fragmentation, as well as a significant modification in the expression levels of genes involved in stress responses, DNA damage recognition and apoptosis pathway. Interestingly, we found that, within the investigated experimental conditions, the toxic effects in the exposed organisms were directly related to the concentration of the AuNPs administered, irrespective of their size.     

Identification of Polymorphisms in the Rabbit Growth Hormone Receptor (GHR) Gene and Association with Finishing Weight in a Commercial Meat Rabbit Line

A shortcut to identify DNA markers associated with economic traits is to use a candidate gene approach that is still useful in livestock species in which molecular tools and resources are not advanced or not well developed. Mutations in the growth hormone receptor (GHR) gene associated with production traits have been already described in several livestock species. For this reason GHR could be an interesting candidate gene in the rabbit. In this study we re-sequenced all exons and non-coding regions of the rabbit GHR gene in a panel of 10 different rabbits and identified 10 single nucleotide polymorphisms (SNPs). One of them (g.63453192C>G or c.106C>G), located in exon 3 was a missense mutation (p.L36V) substituting an amino acid in a highly conserved position across all mammals. This mutation was genotyped in 297 performance tested rabbits of a meat male line and association analysis showed that the investigated SNP was associated with weight at 70 days (P < 0.05). The most frequent genotype (GG) was in animals with higher weight at this age, suggesting that the high directional selection pressure toward this trait since the constitution of the genotyped line might have contributed to shape allele frequencies at this polymorphic site.     

Human Immunodeficiency Virus Type 1 Strains R5 and X4 Induce Different Pathogenic Effects in hu-PBL-SCID Mice, Depending on the State of Activation/Differentiation of Human Target Cells at the Time of Primary Infection

In a previous study, we had found that the extent of T-cell dysfunctions induced by a T-tropic strain of human immunodeficiency virus type 1 (HIV-1) in SCID mice reconstituted with human peripheral blood lymphocytes (hu-PBLs) (hu-PBL-SCID mice) was related to the in vivo state of activation of the human lymphocytes. In this article, we compared the effect of infection of hu-PBL-SCID mice with either T-tropic (X4) or M-tropic (R5) strains of HIV-1 by performing virus inoculation at either 2 h or 2 weeks after the hu-PBL transfer, when the human T cells exhibited a marked activation state or a predominant memory phenotype, respectively. A comparable level of infection was found when hu-PBL-SCID mice were challenged with either the SF162 R5 or the IIIB X4 strain of HIV at 2 h postreconstitution, while at 2 weeks, the R5 virus infection resulted in a higher level of HIV replication than the X4 virus. The R5 strain induced a marked human CD4(+) T-cell depletion along with a drop in levels of human immunoglobulin M in serum and release of soluble factors at both infection times, while the X4 virus induced severe immune dysfunctions only at 2 h. Of interest, injection of hu-PBLs into SCID mice resulted in a marked up-regulation of CCR5 on human CD4(+) T cells. The percentage of CXCR4(+) cells did not change after transplantation, even though a significant decrease in antigen expression was observed. Comparative experiments with two molecular clones of HIV-1 (X4 SF2 and R5 SF162) and two envelope recombinant viruses generated from these viruses showed that R5 viruses (SF162 and the chimeric env-SF162-SF2) caused an extensive depletion of human CD4(+) T cells in SCID mice at both 2 h and 2 weeks after reconstitution, while the X4 viruses (SF2 and the chimeric env-SF2-SF162) induced CD4 T-cell depletion only when infection was performed at the 2-h reconstitution time. These results emphasize the importance of the state of activation/differentiation of human CD4(+) T cells and gp120-coreceptor interactions at the time of primary infection in determining HIV-1 pathogenicity in the hu-PBL-SCID mouse model.     

Antibody Complementarity-Determining Regions (CDRs): A Bridge between Adaptive and Innate Immunity

Background

It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities.

Methodology/Principal Findings

In this study we demonstrate that a synthetic peptide with sequence identical to V_HCDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, V_HCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties.

Conclusions/Significance

These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.     

UVB-radiation-induced apoptosis in Jurkat cells: a coordinated fourier transform infrared spectroscopy-flow cytometry study

We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290-320 nm) at a dose of 310 mJ/ cm2. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis. The behavior of the wave number shift of both the Amide I beta-sheet component and the area of the 1083 cm(-1) band reproduced, with a high correlation, the behavior of the early apoptotic cell population, while the behavior of the Amide I area showed a high correlation with the early plus late apoptotic cell population.     

Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

Conserved Alternative Splicing of Arabidopsis Transthyretin-Like Determines Protein Localization and S-Allantoin Synthesis in Peroxisomes

S-allantoin, a major ureide compound, is produced in plant peroxisomes from oxidized purines. Sequence evidence suggested that the Transthyretin-like (TTL) protein, which interacts with brassinosteroid receptors, may act as a bifunctional enzyme in the synthesis of S-allantoin. Here, we show that recombinant TTL from Arabidopsis thaliana catalyzes two enzymatic reactions leading to the stereoselective formation of S-allantoin, hydrolysis of hydroxyisourate through a C-terminal Urah domain, and decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline through an N-terminal Urad domain. We found that two different mRNAs are produced from the TTL gene through alternative use of two splice acceptor sites. The corresponding proteins differ in the presence (TTL¹⁻) and the absence (TTL²⁻) of a rare internal peroxisomal targeting signal (PTS2). The two proteins have similar catalytic activity in vitro but different in vivo localization: TTL¹⁻ localizes in peroxisomes, whereas TTL²⁻ localizes in the cytosol. Similar splice variants are present in monocots and dicots. TTL originated in green algae through a Urad-Urah fusion, which entrapped an N-terminal PTS2 between the two domains. The presence of this gene in all Viridiplantae indicates that S-allantoin biosynthesis has general significance in plant nitrogen metabolism, while conservation of alternative splicing suggests that this mechanism has general implications in the regulation of the ureide pathway in flowering plants.     

Dopamine-derived quinones affect the structure of the redox sensor DJ-1 through modifications at Cys-106 and Cys-53

The physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced by DAQ adduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1-transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult.     

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.