Genetic assessment of environmental features that influence deer dispersal: implications for prion-infected populations

The landscape can influence host dispersal and density, which in turn, affect infectious disease transmission, spread, and persistence. Understanding how the landscape influences wildlife dispersal and pathogen epidemiology can enhance the efficacy of disease management in natural populations. We applied landscape genetics to examine relationships among landscape variables, dispersal of white-tailed deer hosts and transmission/spread of chronic wasting disease (CWD), a fatal prion encephalopathy. Our focus was on quantifying movements and population structure of host deer in infected areas as a means of predicting the spread of this pathology and promoting its adaptive management. We analyzed microsatellite genotypes of CWD-infected and uninfected deer from two disease foci (Southern Wisconsin, Northern Illinois). We quantified gene flow and population structure using F _ST, assignment tests, and spatial autocorrelation analyses. Gene flow estimates were then contrasted against a suite of landscape variables that potentially mediate deer dispersal. Forest fragmentation and grassland connectivity promoted deer movements while rivers, agricultural fields and large urbanized areas impeded movement. Landscape variables, deer dispersal, and disease transmission covaried significantly and positively in our analyses. Habitats with elevated host gene flow supported the concept of dispersal-mediated CWD transmission by reflecting a concomitant, rapid CWD expansion. Large, interrelated social groups isolated by movement barriers overlapped disease foci, suggesting that philopatry exacerbated CWD transmission. Our results promote adaptive management of CWD by predicting patterns of its spread and identifying habitats at risk for invasion. Further, our landscape genetics approach underscores the significance of topography and host behavior in wildlife disease transmission.     

Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata

Defensins are a large family of small, cysteine‐rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C‐terminal pro‐peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt‐thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC₅₀) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5–10 times less effective at killing necrotrophs, but only two‐fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad‐spectrum resistance to pathogens.     

Florida’s efforts to assume section 404 permitting

Wetlands Ecology and Management - Section 404 of the federal Clean Water Act prohibits disposing of dredged and fill materials into the waters of the United States (WOTUS) absent a permit...     

G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs

Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (Gα-interacting vesicle-associated protein) is the first non-receptor guanine nucleotide exchange factor (GEF) that activates Gα(i) subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for Gα(i) binding and activation. Bioinformatics searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to the GEF sequence of GIV. Using in vitro pulldown and competition assays, we demonstrate that this motif binds preferentially to the inactive conformation of Gα(i1) and Gα(i3) over other Gα subunits and, like GIV, docks onto the α3/switch II cleft. Calnuc binding was impaired when Lys-248 in the α3 helix of Gα(i3) was replaced with M, the corresponding residue in Gα(o), which does not bind to Calnuc. Moreover, mutation of hydrophobic residues in the conserved motif predicted to dock on the α3/switch II cleft of Gα(i3) impaired the ability of Calnuc and NUCB2 to bind and activate Gα(i3) in vitro. We also provide evidence that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gα(i3) in vitro and in cells, probably by inducing a conformational change that renders the Gα(i)-binding residues inaccessible. Taken together, our results identify a new type of Gα(i)-regulatory motif named the GBA motif (for Gα-binding and -activating motif), which is conserved across different proteins throughout evolution. These findings provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα subunits and its regulation by calcium ions.     

Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice

Mammalian pre-mRNA alternative splicing mechanisms are typically studied using artificial minigenes in cultured cells, conditions that may not accurately reflect the physiological context of either the pre-mRNA or the splicing machinery. Here, we describe a strategy to investigate splicing of normal endogenous full-length pre-mRNAs under physiological conditions in live mice. This approach employs antisense vivo-morpholinos (vMOs) to mask cis-regulatory sequences or to disrupt splicing factor expression, allowing functional evaluation of splicing regulation in vivo. We applied this strategy to gain mechanistic insight into alternative splicing events involving exons 2 and 16 (E2 and E16) that control the structure and function of cytoskeletal protein 4.1R. In several mouse tissues, inclusion of E16 was substantially inhibited by interfering with a splicing enhancer mechanism using a target protector morpholino that blocked Fox2-dependent splicing enhancers in intron 16 or a splice-blocking morpholino that disrupted Fox2 expression directly. For E2, alternative 3'-splice site choice is coordinated with upstream promoter use across a long 5'-intron such that E1A splices almost exclusively to the distal acceptor (E2dis). vMOs were used to test the in vivo relevance of a deep intron element previously proposed to determine use of E2dis via a two-step intrasplicing model. Two independent vMOs designed against this intronic regulatory element inhibited intrasplicing, robustly switching E1A splicing to the proximal acceptor (E2prox). This finding strongly supports the in vivo physiological relevance of intrasplicing. vMOs represent a powerful tool for alternative splicing studies in vivo and may facilitate exploration of alternative splicing networks in vivo.     

Sumoylation inhibits alpha-synuclein aggregation and toxicity

Posttranslational modification of proteins by attachment of small ubiquitin-related modifier (SUMO) contributes to numerous cellular phenomena. Sumoylation sometimes creates and abolishes binding interfaces, but increasing evidence points to another role for sumoylation in promoting the solubility of aggregation-prone proteins. Using purified α-synuclein, an aggregation-prone protein implicated in Parkinson's disease that was previously reported to be sumoylated upon overexpression, we compared the aggregation kinetics of unmodified and modified α-synuclein. Whereas unmodified α-synuclein formed fibrils, modified α-synuclein remained soluble. The presence of as little as 10% sumoylated α-synuclein was sufficient to delay aggregation significantly in vitro. We mapped SUMO acceptor sites in α-synuclein and showed that simultaneous mutation of lysines 96 and 102 to arginine significantly impaired α-synuclein sumoylation in vitro and in cells. Importantly, this double mutant showed increased propensity for aggregation and cytotoxicity in a cell-based assay and increased cytotoxicity in dopaminergic neurons of the substantia nigra in vivo. These findings strongly support the model that sumoylation promotes protein solubility and suggest that defects in sumoylation may contribute to aggregation-induced diseases.     

Rescue of HIV-1 broad neutralizing antibody-expressing B cells in 2F5 VH x VL knockin mice reveals multiple tolerance controls

The HIV-1 broadly neutralizing Ab (bnAb) 2F5 has been shown to be poly-/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 V(H) knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. In this study, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 V(H) × V(L) KI mice, and find an even higher degree of tolerance control than observed in the 2F5 V(H) KI strain. Although B cell development was severely impaired in 2F5 V(H) × V(L) KI animals, we demonstrate rescue of their B cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 V(H)/V(L) expression by immature/transitional B cells. Importantly, 7% of rescued B cells retained 2F5 V(H)/V(L) expression and secreted Env-specific mAbs with HIV-1-neutralizing activity. This partial rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B cells in 2F5 V(H) × V(L) KI × Eμ-Bcl-2 transgenic mice and significant (yet modest) enrichment of Env-specific B cells and serum Igs. The rescued 2F5 mAb-producing B cell clones in this study are the first examples, to our knowledge, of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs and provide a starting point for design of strategies aimed at rescuing such B cells.     

Trypanosoma brucei: Two mitogen activated protein kinase kinases are dispensable for growth and virulence of the bloodstream form

Mitogen activated protein kinase cascades function in eukaryotic responses to the environment and stress. Trypanosomatid parasites possess protein kinases with sequences characteristic of kinases in such cascades. In this report we use gene knockouts to demonstrate that two mitogen activated kinase kinase genes, MKK1 (Tb927.3.4860) and MKK5 (Tb927.10.5270), are not essential in the pathogenic bloodstream stage of Trypanosoma brucei, either in vitro or in vivo. Bloodstream forms lacking MKK1 showed decreased growth at 39 °C as compared to the parental line. However, unlike its Leishmania orthologue, T. brucei MKK1 does not appear to play a significant role in flagellar biogenesis.     

Regulatory mechanisms for the development of the migratory phenotype: Roles for photoperiod and the gonad

This article is part of a Special Issue “Energy Balance”.     

RASSF tumor suppressor gene family: Biological functions and regulation

Genetic changes through allelic loss and nucleic acid or protein modifications are the main contributors to loss of function of tumor suppressor proteins. In particular, epigenetic silencing of genes by promoter hypermethylation is associated with increased tumor severity and poor survival. The RASSF (Ras association domain family) family of proteins consists of 10 members, many of which are tumor suppressor proteins that undergo loss of expression through promoter methylation in numerous types of cancers such as leukemia, melanoma, breast, prostate, neck, lung, brain, colorectal and kidney cancers. In addition to their tumor suppressor function, RASSF proteins act as scaffolding agents in microtubule stability, regulate mitotic cell division, modulate apoptosis, control cell migration and cell adhesion, and modulate NFκB activity and the duration of inflammation. The ubiquitous functions of these proteins highlight their importance in numerous physiological pathways. In this review, we will focus on the biological roles of the RASSF family members and their regulation.