Blimp-1-Dependent IL-10 Production by Tr1 Cells Regulates TNF-Mediated Tissue Pathology

Tumor necrosis factor (TNF) is critical for controlling many intracellular infections, but can also contribute to inflammation. It can promote the destruction of important cell populations and trigger dramatic tissue remodeling following establishment of chronic disease. Therefore, a better understanding of TNF regulation is needed to allow pathogen control without causing or exacerbating disease. IL-10 is an important regulatory cytokine with broad activities, including the suppression of inflammation. IL-10 is produced by different immune cells; however, its regulation and function appears to be cell-specific and context-dependent. Recently, IL-10 produced by Th1 (Tr1) cells was shown to protect host tissues from inflammation induced following infection. Here, we identify a novel pathway of TNF regulation by IL-10 from Tr1 cells during parasitic infection. We report elevated Blimp-1 mRNA levels in CD4⁺ T cells from visceral leishmaniasis (VL) patients, and demonstrate IL-12 was essential for Blimp-1 expression and Tr1 cell development in experimental VL. Critically, we show Blimp-1-dependent IL-10 production by Tr1 cells prevents tissue damage caused by IFNγ-dependent TNF production. Therefore, we identify Blimp-1-dependent IL-10 produced by Tr1 cells as a key regulator of TNF-mediated pathology and identify Tr1 cells as potential therapeutic tools to control inflammation.     

Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico

Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue‐1 during 2009, 2010, and 2013 in Florida and dengue‐1 and −2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler‐imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV‐1 and −2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV‐1 (6–14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV‐2 than Ae. albopictus (Ae. aegypti, ∼28%; Ae. albopictus, ∼9%). The dissemination rate with dengue‐2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV‐2 isolate from Puerto Rico. These results are discussed in light of Florida's vulnerability to DENV transmission.     

Ecological correlates and phylogenetic signal of host use in North American unionid mussels

Mussels in the order Unionoida comprise ～75% of the world’s freshwater bivalve species and are free-living apart from a brief larval stage that parasitizes fish. We investigated the relationships among species of North American unionid mussels and their known host fishes from a macroevolutionary perspective to test whether and how ecological and evolutionary factors correlate with patterns of host use. A subset of 69 mussel species was chosen based on data availability regarding their fish host repertoires, phylogenetic relationships, and ecology. Despite the brevity of their parasitic life stages, the mussels conformed to the right-skewed distribution of host specificity typical of parasitic taxa, in which most species are specialists and a few are generalists. Phylogenetic least squares regression models identified affinity for low-gradient and riffle habitats, and colonization of post-glacial watersheds as the best predictors for the number of fish host species per mussel. However, the second-best model identified citation number as a predictor of the number of hosts, implying that many mussel–host interactions still remain to be identified. A Multiple Regression Mantel test was performed to identify factors associated with the proportion of hosts shared between pairs of mussel species. Range overlap, citations, genetic distance, and similarity in host infection strategy were significantly correlated with the proportion of hosts shared, yet total variation as explained by the best model was low (R²?=?0.14). There was evidence of a topological association between mussels and their hosts (P?=?0.001) and a significant phylogenetic signal of host specificity (λ?=?0.81, P?=?0.003), indicating closely related mussels that overlap in range are more likely to be competing for hosts. Our results provide an initial macroevolutionary framework for studying the evolution of host infection strategies in these mussels but also highlights gaps still remaining in our fundamental ecological knowledge of this endangered clade.     

An effective method for ecosystem‐scale manipulation of bird abundance and species richness

Manipulation experiments are a cornerstone of ecological research, but can be logistically challenging to execute—particularly when they are intended to isolate the ecological role of large, vagile species, like birds. Despite indirect evidence that birds are influential in many ecosystems, large‐scale, multi‐year bird manipulation experiments are rare. When these studies are conducted, they are typically realized with caged or netted exclosures, an approach that can be expensive, risky for wildlife, and difficult to maintain. In cases where caged exclosures are not appropriate, alternate approaches are needed to allow rigorous empirical studies on the ecological role of birds. Here, we present and validate a method for experimentally increasing the abundance and richness of birds at the scale of entire aquatic ecosystems. Unlike bird exclusion, this approach is experimentally tractable, appealing to land managers, and possible to deploy over large spatial scales. We tested the efficacy of our approach for increasing bird abundance and species richness at 16 central California ponds. Based on bird visitation data obtained by summer camera trapping, our approach significantly increased bird species richness and abundance at manipulated ponds compared to control ponds. Attractant treatments mitigated the negative effects of a major drought on bird species richness and generated a near doubling of bird abundance in the presence of attractants. Treatments had no effect on most mammal species, with the exception of ground squirrels, which increased in abundance in the presence of attractants. These results suggest that attractants are effective in increasing bird abundance and richness. We encourage researchers to consider this approach for experimentally isolating the ecological role of birds in aquatic and open terrestrial ecosystems, especially in cases where cost or logistical constraints preclude the use of caged or netted exclosures.     

Role of the MexAB-OprM efflux pump of Pseudomonas aeruginosa in tolerance to tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and alpha-terpineol

Using a series of efflux mutants of Pseudomonas aeruginosa, the MexAB-OprM pump was identified as contributing to this organism's tolerance to the antimicrobial agent tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and alpha-terpineol. These data show that a multidrug efflux system of P. aeruginosa can extrude monoterpenes and related alcohols.     

Amino Acid Transporters and Exchangers from the SLC1A Family: Structure,Mechanism and Roles in Physiology and Cancer

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems—the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues Glt_Ph and Glt_Tk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

     

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

Alteration of NCoR Corepressor Splicing in Mice Causes Increased Body Weight and Hepatosteatosis without Glucose Intolerance

Alternative mRNA splicing is an important means of diversifying function in higher eukaryotes. Notably, both NCoR and SMRT corepressors are subject to alternative mRNA splicing, yielding a series of distinct corepressor variants with highly divergent functions. Normal adipogenesis is associated with a switch in corepressor splicing from NCoRω to NCoRδ, which appears to help regulate this differentiation process. We report here that mimicking this development switch in mice by a splice-specific whole-animal ablation of NCoRω is very different from a whole-animal or tissue-specific total NCoR knockout and produces significantly enhanced weight gain on a high-fat diet. Surprisingly, NCoRω^−/− mice are protected against diet-induced glucose intolerance despite enhanced adiposity and the presence of multiple additional, prodiabetic phenotypic changes. Our results indicate that the change in NCoR splicing during normal development both helps drive normal adipocyte differentiation and plays a key role in determining a metabolically appropriate storage of excess calories. We also conclude that whole-gene “knockouts” fail to reveal how important gene products are customized, tailored, and adapted through alternative mRNA splicing and thus do not reveal all the functions of the protein products of that gene.     

Ultrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri

Shigella flexneri is a Gram-negative enteric pathogen that is the predominant cause of bacillary dysentery. Shigella uses a type III secretion system to deliver effector proteins that alter normal target cell functions to promote pathogen invasion. The type III secretion apparatus (T3SA) consists of a basal body, an extracellular needle, and a tip complex that is responsible for delivering effectors into the host cell cytoplasm. IpaD [Ipa (invasion plasmid antigen)] is the first protein to localize to the T3SA needle tip, where it prevents premature effector secretion and serves as an environmental sensor for triggering recruitment of the translocator protein IpaB to the needle tip. Thus, IpaD would be expected to form a stable structure whose overall architecture supports its functions. It is not immediately obvious from the published IpaD crystal structure (Protein Data Bank ID 2j0o) how a multimer of IpaD would be incorporated at the tip of the first static T3SA intermediate, nor what its functional role would be in building a mature T3SA. Here, we produce three-dimensional reconstructions from transmission electron microscopy images of IpaD localized at the Shigella T3SA needle tip for comparison to needle tips from a Shigella ipaD-null mutant. The results demonstrate that IpaD resides as a homopentamer at the needle tip of the T3SA. Furthermore, comparison to tips assembled from the distal domain IpaD(Δ192-267) mutation shows that IpaD adopts an elongated conformation that facilitates its ability to control type III secretion and stepwise assembly of the T3SA needle tip complex.