Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments

Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated whether two Caenorhabditis elegans α- and β-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length.     

Post-pandemic seroprevalence of pandemic influenza A (H1N1) 2009 infection (swine flu) among children <18 years in Germany

Background

We determined antibodies to the pandemic influenza A (H1N1) 2009 virus in children to assess: the incidence of (H1N1) 2009 infections in the 2009/2010 season in Germany, the proportion of subclinical infections and to compare titers in vaccinated and infected children.

Methodology/Principal Findings

Eight pediatric hospitals distributed over Germany prospectively provided sera from in- or outpatients aged 1 to 17 years from April 1^st to July 31^st 2010. Vaccination history, recall of infections and sociodemographic factors were ascertained. Antibody titers were measured with a sensitive and specific in-house hemagglutination inhibition test (HIT) and compared to age-matched sera collected during 6 months before the onset of the pandemic in Germany. We analyzed 1420 post-pandemic and 300 pre-pandemic sera. Among unvaccinated children aged 1–4 and 5–17 years the prevalence of HI titers (≥1∶10) was 27.1% (95% CI: 23.5–31.3) and 53.5% (95% CI: 50.9–56.2) compared to 1.7% and 5.5%, respectively, for pre-pandemic sera, accounting for a serologically determined incidence of influenza A (H1N1) 2009 during the season 2009/2010 of 25,4% (95% CI : 19.3–30.5) in children aged 1–4 years and 48.0% (95% CI: 42.6–52.0) in 5–17 year old children. Of children with HI titers ≥1∶10, 25.5% (95% CI: 22.5–28.8) reported no history of any infectious disease since June 2009. Among vaccinated children, 92% (95%-CI: 87.0–96.6) of the 5–17 year old but only 47.8% (95%-CI: 33.5–66.5) of the 1–4 year old children exhibited HI titers against influenza A virus (H1N1) 2009.

Conclusion

Serologically determined incidence of influenza A (H1N1) 2009 infections in children indicates high infection rates with older children (5–17 years) infected twice as often as younger children. In about a quarter of the children with HI titers after the season 2009/2010 subclinical infections must be assumed. Low HI titers in young children after vaccination with the AS03_B-adjuvanted split virion vaccine need further scrutiny.     

A genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Trypanosoma brucei reveals a trypanosome-specific pattern of rRNA modification

Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which function in guiding modifications such as 2'-O-ribose methylation and pseudouridylation on rRNAs and snRNAs. To investigate the genome organization of Trypanosoma brucei snoRNAs and the pattern of rRNA modifications, we used a whole-genome approach to identify the repertoire of these guide RNAs. Twenty-one clusters encoding for 57 C/D snoRNAs and 34 H/ACA-like RNAs, which have the potential to direct 84 methylations and 32 pseudouridines, respectively, were identified. The number of 2'-O-methyls (Nms) identified on rRNA represent 80% of the expected modifications. The modifications guided by these RNAs suggest that trypanosomes contain many modifications and guide RNAs relative to their genome size. Interestingly, approximately 40% of the Nms are species-specific modifications that do not exist in yeast, humans, or plants, and 40% of the species-specific predicted modifications are located in unique positions outside the highly conserved domains. Although most of the guide RNAs were found in reiterated clusters, a few single-copy genes were identified. The large repertoire of modifications and guide RNAs in trypanosomes suggests that these modifications possibly play a central role in these parasites.     

Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.     

Inhibition of HIV-1 envelope glycoprotein-mediated cell fusion by a DL-amino acid-containing fusion peptide: possible recognition of the fusion complex

The N-terminal fusion peptide (FP) of human immunodeficiency virus-1 (HIV-1) is a potent inhibitor of cell-cell fusion, possibly because of its ability to recognize the corresponding segments inside the fusion complex within the membrane. Here we show that a fusion peptide in which the highly conserved Ile(4), Phe(8), Phe(11), and Ala(14) were replaced by their d-enantiomers (IFFA) is a potent inhibitor of cell-cell fusion. Fourier transform infrared spectroscopy confirmed that despite these drastic modifications, the peptide preserved most of its structure within the membrane. Fluorescence energy transfer studies demonstrated that the diastereomeric peptide interacted with the wild type FP, suggesting this segment as the target site for inhibition of membrane fusion. This is further supported by the similar localization of the wild type and IFFA FPs to microdomains in T cells and the preferred partitioning into ordered regions within sphingomyelin/phosphatidyl-choline/cholesterol giant vesicles. These studies provide insight into the mechanism of molecular recognition within the membrane milieu and may serve in designing novel HIV entry inhibitors.     

A new group of antifungal and antibacterial lipopeptides derived from non-membrane active peptides conjugated to palmitic acid

We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing d- and l-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K(4)X(7)W, where X designates Gly, Ala, Val, or Leu (designated d-X peptides). The data revealed that PA-d-G and PA-d-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-d-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both d-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-d-L and PA-d-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections.     

Body size of insular carnivores: little support for the island rule

Large mammals are thought to evolve to be smaller on islands, whereas small mammals grow larger. A negative correlation between relative size of island individuals and body mass is termed the "island rule." Several mechanisms--mainly competitive release, resource limitation, dispersal ability, and lighter predation pressure on islands, as well as a general physiological advantage of modal size--have been advanced to explain this pattern. We measured skulls and teeth of terrestrial members of the order Carnivora in order to analyze patterns of body size evolution between insular populations and their near mainland conspecifics. No correlations were found between the size ratios of insular/mainland carnivore species and body mass. Only little support for the island rule is found when individual populations rather than species are considered. Our data are at odds with those advanced in support of theories of optimal body size. Carnivore size is subjected to a host of selective pressures that do not vary uniformly from place to place. Mass alone cannot account for the patterns in body size of insular carnivores.     

Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins

Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-l and all-d amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-d specifically associated with Tar-1 all-l within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-l and all-d, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-l and all-d showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-d TM domain peptide acts in vivo similarly to its parental all-l peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides.     

Extraordinary resistance to insecticides reveals exotic Q biotype of Bemisia tabaci in the New World

A strain of the whitefly Bemisia tabaci (Gennadius) possessing unusually high levels of resistance to a wide range of insecticides was discovered in 2004 in the course of routine resistance monitoring in Arizona. The multiply resistant insects, collected from poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) plants purchased at a retail store in Tucson, were subjected to biotype analysis in three laboratories. Polyacrylamide gel electrophoresis of naphthyl esterases and sequencing of the mitochondrial cytochrome oxidase I gene (780 bp) confirmed the first detection of the Q biotype of B. tabaci in the New World. This U.S. Q biotype strain, referred to as Poinsettia'04, was highly resistant to two selective insect growth regulators, pyriproxyfen and buprofezin, and to mixtures of fenpropathrin and acephate. It was also unusually low in susceptibility to the neonicotinoid insecticides imidacloprid, acetamiprid, and thiamethoxam, relative to B biotype whiteflies. In 100 collections of whiteflies made in Arizona cotton (Gossypium spp.), vegetable, and melon (Cucumis melo L.) fields from 2001 to 2005, no Q biotypes were detected. Regions of the United States that were severely impacted by the introduction of the B biotype of B. tabaci in the 1980s would be well advised to promote measures that limit movement of the Q biotype from controlled environments into field systems and to formulate alternatives for managing this multiply-resistant biotype, in the event that it becomes more widely distributed.