Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO₄ backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.     

Identification of Tenrec ecaudatus,a Wild Mammal Introduced to Mayotte Island,as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis

Leptospirosis is a bacterial zoonosis of major concern on tropical islands. Human populations on western Indian Ocean islands are strongly affected by the disease although each archipelago shows contrasting epidemiology. For instance, Mayotte, part of the Comoros Archipelago, differs from the other neighbouring islands by a high diversity of Leptospira species infecting humans that includes Leptospira mayottensis, a species thought to be unique to this island. Using bacterial culture, molecular detection and typing, the present study explored the wild and domestic local mammalian fauna for renal carriage of leptospires and addressed the genetic relationships of the infecting strains with local isolates obtained from acute human cases and with Leptospira strains hosted by mammal species endemic to nearby Madagascar. Tenrec (Tenrec ecaudatus, Family Tenrecidae), a terrestrial mammal introduced from Madagascar, is identified as a reservoir of L. mayottensis. All isolated L. mayottensis sequence types form a monophyletic clade that includes Leptospira strains infecting humans and tenrecs on Mayotte, as well as two other Malagasy endemic tenrecid species of the genus Microgale. The lower diversity of L. mayottensis in tenrecs from Mayotte, compared to that occurring in Madagascar, suggests that L. mayottensis has indeed a Malagasy origin. This study also showed that introduced rats (Rattus rattus) and dogs are probably the main reservoirs of Leptospira borgpetersenii and Leptospira kirschneri, both bacteria being prevalent in local clinical cases. Data emphasize the epidemiological link between the two neighbouring islands and the role of introduced small mammals in shaping the local epidemiology of leptospirosis.     

Intravascular magnetomotive optical coherence tomography of targeted early‐stage atherosclerotic changes in ex vivo hyperlipidemic rabbit aortas

We report the development of an intravascular magnetomotive optical coherence tomography (IV‐MM‐OCT) system used with targeted protein microspheres to detect early‐stage atherosclerotic fatty streaks/plaques. Magnetic microspheres (MSs) were injected in vivo in rabbits, and after 30 minutes of in vivo circulation, excised ex vivo rabbit aorta samples specimens were then imaged ex vivo with our prototype IV‐MM‐OCT system. The alternating magnetic field gradient was provided by a unique pair of external custom‐built electromagnetic coils that modulated the targeted magnetic MSs. The results showed a statistically significant MM‐OCT signal from the aorta samples specimens injected with targeted MSs.

Representative magnetomotive signal (green) using targeted and non‐targeted magnetomotive microspheres in atherosclerotic diseased rabbit aortas.     

Comparison of native and non‐native ubiquitin oligomers reveals analogous structures and reactivities

Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post‐translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non‐enzymatic methods to synthesize Ub chains of well‐defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non‐native linkages; however, few studies have examined the extent to which non‐native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we compare the structure and function of Ub dimers bearing native and non‐native linkages. Using small‐angle X‐ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non‐native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady‐state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non‐native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non‐native linkages can serve as surrogate substrates for explorations of Ub function.     

A higher-order entity formed by the flexible assembly of RAP1 with TRF2

Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments.     

Integrity of mating behaviors and seasonal reproduction in coyotes (Canis latrans) following treatment with estradiol benzoate

Coyotes (Canis latrans) are seasonally monestrous and form perennial pair-bonds. Breeding is dominated by each pack's alpha male and female, and both sexes share responsibility for territory defense and pup-rearing. They are also opportunistic predators on domestic livestock and pets. But while dominant adults have been implicated as primary killers, depredation is reduced when coyotes are without pups. Contraception, therefore, may represent a non-lethal solution for conflicts between coyotes and humans. Steroid hormones successfully control fertility in some species, but have been considered contraindicated in wildlife and canids in particular; specific concerns include possible induction of aberrant behavior, or uterine and hematopoietic pathologies. Herein we describe a study examining the physiological effectiveness, health safety, and behavioral consequences following treatment of estrous coyotes with exogenous estrogen. We treated captive adult female coyotes in estrus with 0.01 mg/kg estradiol benzoate (EB), either before (n = 5) or immediately after ovulation (n = 6), then documented reproductive outcome, physiological variables and behavioral responses, during and after treatment. Pregnancy was averted in six females treated after ovulation, suggesting that appropriate timing of treatment proved crucial. A transient suppression of sexual behavior was observed, and in some cases, estrus appeared slightly lengthened. However, neither ovulation nor mating behavior was fully suppressed. Importantly, non-pregnant females (and their mates) displayed diestrous socio-sexual behavior similar to pregnant coyotes (behavioral pseudopregnancy). Furthermore, non-pregnant coyotes did not mate again until the next native breeding season, and we observed no deleterious physiological effects during diestrus or subsequent ovarian cycles.     

Hydrogen peroxide is the major oxidant product of xanthine oxidase

Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) in inflammatory disease. Focus, however, has centered almost exclusively on XO-derived superoxide (O₂^??), whereas direct H₂O₂ production from XO has been less well investigated. Therefore, we examined the relative quantities of O₂^?? and H₂O₂ produced by XO under a range (1–21%) of O₂ tensions. At O₂ concentrations between 10 and 21%, H₂O₂ accounted for ～75% of ROS production. As O₂ concentrations were lowered, there was a concentration-dependent increase in H₂O₂ formation, accounting for 90% of ROS production at 1% O₂. Alterations in pH between 5.5 and 7.4 did not affect the relative proportions of H₂O₂ and O₂^?? formation. Immobilization of XO, by binding to heparin–Sepharose, further enhanced relative H₂O₂ production by ～30%, under both normoxic and hypoxic conditions. Furthermore, XO bound to glycosaminoglycans on the apical surface of bovine aortic endothelial cells demonstrated a similar ROS production profile. These data establish H₂O₂ as the dominant (70–95%) reactive product produced by XO under clinically relevant conditions and emphasize the importance of H₂O₂ as a critical factor when examining the contributory roles of XO-catalyzed ROS in inflammatory processes as well as cellular signaling.     

DIRS1 -like retrotransposons are widely distributed among Decapoda and are particularly present in hydrothermal vent organisms

Background  

Transposable elements are major constituents of eukaryote genomes and have a great impact on genome structure and stability. Considering their mutational abilities, TEs can contribute to the genetic diversity and evolution of organisms. Knowledge of their distribution among several genomes is an essential condition to study their dynamics and to better understand their role in species evolution. DIRS1 -like retrotransposons are a particular group of retrotransposons according to their mode of transposition that implies a tyrosine recombinase. To date, they have been described in a restricted number of species in comparison with the LTR retrotransposons. In this paper, we determine the distribution of DIRS1 -like elements among 25 decapod species, 10 of them living in hydrothermal vents that correspond to particularly unstable environments.     

Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed 3–11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirB6 were consistently predicted, and B. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-α and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp38 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4⁺ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88^−/− mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive interferon-γ-dependent CD4⁺ T cell polarization and antibody switching.Human filariasis is a major neglected tropical disease. More than 150 million individuals are infected with the filarial worms responsible for lymphatic filariasis (LF)⁴ (Wuchereria bancrofti and Brugia malayi) and onchocerciasis (Onchocerca volvulus). Over 40 million suffer from disfiguring and incapacitating disease with an estimated 1.5 billion people at risk of infection, ranking filariasis as one of the major causes of global morbidity (1).A feature of filarial pathogenesis is a host inflammatory response provoked by the death of larvae and adult stages within parasitized tissues (2). All causative agents of LF and O. volvulus harbor an intracellular symbiotic bacterium, Wolbachia, and are reliant on this endosymbiont for embryogenesis, growth, and survival (3). Previous studies have determined that the inflammatory potential of B. malayi and O. volvulus is dependent on the presence of Wolbachia. For example, Wolbachia-containing filarial extracts induce activation and tolerance in murine macrophages (4, 5), activate human monocytes (6), and activate human and murine neutrophils (7, 8). In addition, O. volvulus and B. malayi extracts containing Wolbachia stimulate neutrophil recruitment to the corneal stroma and development of corneal haze in a murine model of ocular onchocerciasis, in contrast with an aposymbiotic filaria (9). Moreover, isolated Wolbachia from filaria or from insect cells can replicate these effects (8, 10). The activation of neutrophils results in further neutrophil recruitment leading to the disruption of normal corneal clarity and development of stromal haze (11).Activation and subsequent desensitization of macrophages by Wolbachia molecules has been shown to be dependent on TLR2 and the adaptor molecule MyD88 (5, 10). Further studies have established that Wolbachia-induced inflammation is dependent on TLR2 and TLR6 recognition and signaling through the MyD88/Mal pathway and are independent of TRIF and TRAM (12). However, Wolbachia ligands for TLR2/TLR6 have not been characterized. To address this, we used the TLR receptor recognition profile to identify TLR2/6 ligands in the Wolbachia genome. In this study, we demonstrate that Wolbachia-derived diacyl-lipoproteins are candidate stimulatory molecules required for TLR2/6 ligation and production of pro-inflammatory cytokine and chemokine responses. Furthermore, we show that a synthetic Wolbachia lipopeptide (Diacyl WoLP) induces TLR2/6-dependent corneal inflammation, and TLR2-dependent TNFα responses in filarial disease models and up-regulates surface markers of human lymphatic endothelium. Diacyl WoLP also induced activation and maturation of dendritic cells and generated type 1 CD4⁺ T cell and antibody responses to filarial antigens.