Reproductive costs to male and female worm pipefish

Prior to reproductive maturity male and female worm pipefish Nerophis lumbriciformis are not significantly different in standard length ( L _S), eviscerated mass or condition indices. Subsequently, however, mature females achieve significantly ( P  < 0·001) greater values of L _S and mass. For egg-bearing males, there was no marked difference in the mass of embryos with increasing development ( P  > 0·1); similarly there was no significant divergence in condition indices calculated for males carrying embryos of increasing development. Post-brooding males, however, had significantly lower values for the hepato-somatic index than either egg-bearing or non-mated male worm pipefish ( P  < 0·001). This study suggests that male worm pipefish invest a significant proportion of their energy budget into the development and maintenance of the brooding patch, to the detriment of increased length or mass.     

MyD88 Deficiency Markedly Worsens Tissue Inflammation and Bacterial Clearance in Mice Infected with Treponema pallidum,the Agent of Syphilis

Research on syphilis, a sexually transmitted infection caused by the non-cultivatable spirochete Treponema pallidum, has been hampered by the lack of an inbred animal model. We hypothesized that Toll-like receptor (TLR)-dependent responses are essential for clearance of T. pallidum and, consequently, compared infection in wild-type (WT) mice and animals lacking MyD88, the adaptor molecule required for signaling by most TLRs. MyD88-deficient mice had significantly higher pathogen burdens and more extensive inflammation than control animals. Whereas tissue infiltrates in WT mice consisted of mixed mononuclear and plasma cells, infiltrates in MyD88-deficient animals were predominantly neutrophilic. Although both WT and MyD88-deficient mice produced antibodies that promoted uptake of treponemes by WT macrophages, MyD88-deficient macrophages were deficient in opsonophagocytosis of treponemes. Our results demonstrate that TLR-mediated responses are major contributors to the resistance of mice to syphilitic disease and that MyD88 signaling and FcR-mediated opsonophagocytosis are linked to the macrophage-mediated clearance of treponemes.     

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition

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Probing Neuroserpin Polymerization and Interaction with Amyloid-β Peptides Using Single Molecule Fluorescence

Neuroserpin is a member of the serine proteinase inhibitor superfamily. It can undergo a conformational transition to form polymers that are associated with the dementia familial encephalopathy with neuroserpin inclusion bodies and the wild-type protein can inhibit the toxicity of amyloid-β peptides in Alzheimer's disease. We have used a single molecule fluorescence method, two color coincidence detection, to determine the rate-limiting steps of the early stages of the polymerization of fluorophore-labeled neuroserpin and have assessed how this process is altered in the presence of Aβ_1-40. Our data show that neuroserpin polymerization proceeds first by the unimolecular formation of an active monomer, followed by competing processes of both polymerization and formation of a latent monomer from the activated species. These data are not in keeping with the recently proposed domain swap model of polymer formation in which the latent species and activated monomer are likely to be formed by competing pathways directly from the unactivated monomeric serpin. Moreover, the Aβ_1-40 peptide forms a weak complex with neuroserpin (dissociation constant of 10 ± 5 nM) that increases the amount of active monomer thereby increasing the rate of polymerization. The Aβ_1-40 is displaced from the complex so that it acts as a catalyst and is not incorporated into neuroserpin polymers.     

DySCo: Quantitating Associations of Membrane Proteins Using Two-Color Single-Molecule Tracking

We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small.     

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Background

Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.

Methodology/Principal Findings

A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals'' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.

Conclusion/Significance

This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.     

AT1 Angiotensin Receptors Mobilize Intracellular Calcium in a Subclone of NG108–15 Neuroblastoma Cells

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro efficacy, resistance selection, and structural modeling studies implicate the malarial parasite apicoplast as the target of azithromycin

Azithromycin (AZ), a broad-spectrum antibacterial macrolide that inhibits protein synthesis, also manifests reasonable efficacy as an antimalarial. Its mode of action against malarial parasites, however, has remained undefined. Our in vitro investigations with the human malarial parasite Plasmodium falciparum document a remarkable increase in AZ potency when exposure is prolonged from one to two generations of intraerythrocytic growth, with AZ producing 50% inhibition of parasite growth at concentrations in the mid to low nanomolar range. In our culture-adapted lines, AZ displayed no synergy with chloroquine (CQ), amodiaquine, or artesunate. AZ activity was also unaffected by mutations in the pfcrt (P. falciparum chloroquine resistance transporter) or pfmdr1 (P. falciparum multidrug resistance-1) drug resistance loci, as determined using transgenic lines. We have selected mutant, AZ-resistant 7G8 and Dd2 parasite lines. In the AZ-resistant 7G8 line, the bacterial-like apicoplast large subunit ribosomal RNA harbored a U438C mutation in domain I. Both AZ-resistant lines revealed a G76V mutation in a conserved region of the apicoplast-encoded P. falciparum ribosomal protein L4 (PfRpl4). This protein is predicted to associate with the nuclear genome-encoded P. falciparum ribosomal protein L22 (PfRpl22) and the large subunit rRNA to form the 50 S ribosome polypeptide exit tunnel that can be occupied by AZ. The PfRpl22 sequence remained unchanged. Molecular modeling of mutant PfRpl4 with AZ suggests an altered orientation of the L75 side chain that could preclude AZ binding. These data imply that AZ acts on the apicoplast bacterial-like translation machinery and identify Pfrpl4 as a potential marker of resistance.     

Influence of Exposure History on the Immunology and Development of Resistance to Human Schistosomiasis Mansoni

Background

Previous studies suggest that humans can acquire immunity to reinfection with schistosomes, most probably due to immunologic mechanisms acquired after exposure to dying schistosome worms.

Methodology/Principal Findings

We followed longitudinally two cohorts of adult males occupationally exposed to Schistosoma mansoni by washing cars (120 men) or harvesting sand (53 men) in Lake Victoria. Men were treated with praziquantel each time S. mansoni infection was detected. In car washers, a significant increase in resistance to reinfection, as measured by the number of cars washed between cure and reinfection, was observed after the car washers had experienced, on average, seven cures. In the car washers who developed resistance, the level of schistosome-specific IgE increased between baseline and the time at which development of resistance was first evidenced. In the sand harvesters, a significant increase in resistance, as measured by the number of days worked in the lake between cure and reinfection, was observed after only two cures. History of exposure to S. mansoni differed between the two cohorts, with the majority of sand harvesters being lifelong residents of a village endemic for S. mansoni and the majority of car washers having little exposure to the lake before they began washing cars. Immune responses at study entry were indicative of more recent infections in car washers and more chronic infections in sand harvesters.

Conclusions/Significance

Resistance to reinfection with S. mansoni can be acquired or augmented by adults after multiple rounds of reinfection and cure, but the rate at which resistance is acquired by this means depends on immunologic status and history of exposure to S. mansoni infection.