Aspirin treatment of mice infected with Trypanosoma cruzi and implications for the pathogenesis of Chagas disease

Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved ejection fraction. ASA treatment diminished the profile of parasite- and host-derived circulating prostaglandins in infected mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1) null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane (TX)A(2) and prostaglandin (PG)F(2α). Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in ASA-treated infected mice indicating the effects of ASA on mortality potentially had little to do with inhibition of prostaglandin metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNFα reduced, in the spleens of infected ASA-treated mice. Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely succumbs more quickly without the initiation of the "cytokine storm" during acute infection. We conclude that ASA, through both COX inhibition and other "off-target" effects, modulates the progression of acute and chronic Chagas disease. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences between host response in the acute and chronic T. cruzi infection.     

Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies

Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies.     

Empowering international canine inherited disorder management

The mapping of the canine genome and the study of canine breed genomic architecture has revolutionized the discovery of genetic tests for inherited disorders in dogs. As the genetics underlying complex disorders are revealed, canine breeders and their registering organisations will be required to understand genetics in a much more sophisticated way. To facilitate the management of genetic disorders in the era of new complex information, we consider how best to apply the results of new research and analytical techniques to benefit the wider canine breeding community with the aims of improving canine health and maintaining benevolent genetic diversity. If this is not done, there is a serious risk that expensive and valuable genetic research will remain unused or be misused to the detriment of breeds. In this review, we make a case for the formation of an international organisation that will exist as a central repository for breed-based genetic analysis and information sharing. This organisation ("Inter-Dog") could be modelled on a similar organisation that is monitoring genetic improvement of dairy cattle. The formation of such an organisation will require the collaboration of international kennel management organisations, researchers, and agencies offering genetic testing services.     

Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor signaling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin signaling. We identified activated leukocyte cell adhesion molecule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neurotrophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75(NTR) toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system.     

A one dose experimental cholera vaccine

The number of cholera vaccine doses required for immunity is a constraint during epidemic cholera. Protective immunity following one dose of multiple Vibrio cholerae (Vc) colonization factors (Inaba LPS El Tor, TcpA, TcpF, and CBP-A) has not been directly tested even though individual Vc colonization factors are the protective antigens. Inaba LPS consistently induced vibriocidal and protective antibodies at low doses. A LPS booster, regardless of dose, induced highly protective secondary sera. Vc protein immunogens emulsified in adjuvant were variably immunogenic. CBP-A was proficient at inducing high IgG serum titers compared with TcpA or TcpF. After one immunization, TcpA or TcpF antisera protected only when the toxin co-regulated pilus operon of the challenge Vc was induced by AKI culture conditions. CBP-A was not consistently able to induce protection independent of the challenge Vc culture conditions. These results reveal the need to understand how best to leverage the 'right' Vc immunogens to obtain durable immunity after one dose of a cholera subunit vaccine. The dominance of the protective anti-LPS antibody response over other Vc antigen antibody response needs to be controlled to find other protective antigens that can add to anti-LPS antibody-based immunity.     

Predators alter the scaling of diversity in prey metacommunities

Although predator effects on the number of locally coexisting species are well understood, there are few formal predictions of how these local predator effects influence patterns of prey diversity at larger spatial scales. Building on the theory of island biogeography, we develop a simple model that describes how predators can alter the scaling of diversity in prey metacommunities and compares the effects of generalist and specialist predators on regional prey diversity. Generalist predators, which consume prey randomly with respect to species identity, are predicted to reduce α‐diversity and increase β‐diversity thereby maintaining regional diversity (γ‐diversity). Alternatively, specialist predators, which filter out prey species intolerant of predators, are predicted to reduce bothα‐diversity andβ‐diversity by causing the same prey species to be extirpated in each locality, resulting in regional prey species extinctions and lower γ‐diversity. These distinct effects of generalist and specialist predators on prey diversity at different spatial scales are uniquely shaped by the extent of predation within those metacommunities. Overall, our model results make general predictions for how different types of predators can differentially affect prey diversity across spatial scales, allowing a more complete understanding of the possible implications of predator eradications or introductions for biodiversity.     

Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance. Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation phenotype due to Tet1 depletion positively correlated with the extent of 5hmC loss. Meta-analyses of genomic data sets suggested interaction between Tet1 and leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated differentiation. Withdrawal of LIF leads to differentiation of mESCs. We discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene activation by affecting Stat3's ability to bind to its target sites on chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further supporting the dependence of LIF/Stat3 signaling on Tet1. These data support the conclusion that analysis of mESCs in the hours/days immediately following efficient Tet1 depletion reveals Tet1's normal physiological role in maintaining the pluripotent state that may be subject to homeostatic compensation in genetic models.     

Human tRNA genes function as chromatin insulators

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes.