Patterns of livestock depredation and cost‐effectiveness of fortified livestock enclosures in northern Tanzania

Human–carnivore conflicts and retaliatory killings contribute to carnivore populations' declines around the world. Strategies to mitigate conflicts have been developed, but their efficacy is rarely assessed in a randomized case–control design. Further, the economic costs prevent the adoption and wide use of conflict mitigation strategies by pastoralists in rural Africa. We examined carnivore (African lion [Panthera leo], leopard [Panthera pardus], spotted hyena [Crocuta crocuta], jackal [Canis mesomelas], and cheetah [Acinonyx jubatus]) raids on fortified (n = 45, total 631 monthly visits) and unfortified (traditional, n = 45, total 521 monthly visits) livestock enclosures (“bomas”) in northern Tanzania. The study aimed to (a) assess the extent of retaliatory killings of major carnivore species due to livestock depredation, (b) describe the spatiotemporal characteristics of carnivore raids on livestock enclosures, (c) analyze whether spatial covariates influenced livestock depredation risk in livestock enclosures, and (d) examine the cost‐effectiveness of livestock enclosure fortification. Results suggest that (a) majority of boma raids by carnivores were caused by spotted hyenas (nearly 90% of all raids), but retaliatory killings mainly targeted lions, (b) carnivore raid attempts were rare at individual households (0.081 raid attempts/month in fortified enclosures and 0.102 raid attempts/month in unfortified enclosures), and (c) spotted hyena raid attempts increased in the wet season compared with the dry season, and owners of fortified bomas reported less hyena raid attempts than owners of unfortified bomas. Landscape and habitat variables tested, did not strongly drive the spatial patterns of spotted hyena raids in livestock bomas. Carnivore raids varied randomly both spatially (village to village) and temporally (year to year). The cost‐benefit analysis suggest that investing in boma fortification yielded positive net present values after two to three years. Thus, enclosure fortification is a cost‐effective strategy to promote coexistence of carnivores and humans.     

Accurate Blood Flow Measurements: Are Artificial Tracers Necessary?

Imaging-based blood flow measurement techniques, such as particle image velocimetry, have become an important tool in cardiovascular research. They provide quantitative information about blood flow, which benefits applications ranging from developmental biology to tumor perfusion studies. Studies using these methods can be classified based on whether they use artificial tracers or red blood cells to visualize the fluid motion. We here present the first direct comparison in vivo of both methods. For high magnification cases, the experiments using red blood cells strongly underestimate the flow (up to 50% in the present case), as compared to the tracer results. For medium magnification cases, the results from both methods are indistinguishable as they give the same underestimation of the real velocities (approximately 33%, based on in vitro reference measurements). These results suggest that flow characteristics reported in literature cannot be compared without a careful evaluation of the imaging characteristics. A method to predict the expected flow averaging behavior for a particular facility is presented.     

The Light Chain 1 Subunit of the Microtubule-Associated Protein 1B (MAP1B) Is Responsible for Tiam1 Binding and Rac1 Activation in Neuronal Cells

Microtubule-associated protein 1B (MAP1B) is a neuronal protein involved in the stabilization of microtubules both in the axon and somatodendritic compartments. Acute, genetic inactivation of MAP1B leads to delayed axonal outgrowth, most likely due to changes in the post-translational modification of tubulin subunits, which enhances microtubule polymerization. Furthermore, MAP1B deficiency is accompanied by abnormal actin microfilament polymerization and dramatic changes in the activity of small GTPases controlling the actin cytoskeleton. In this work, we showed that MAP1B interacts with a guanine exchange factor, termed Tiam1, which specifically activates Rac1. These proteins co-segregated in neurons, and interact in both heterologous expression systems and primary neurons. We dissected the molecular domains involved in the MAP1B-Tiam1 interaction, and demonstrated that pleckstrin homology (PH) domains in Tiam1 are responsible for MAP1B binding. Interestingly, only the light chain 1 (LC1) of MAP1B was able to interact with Tiam1. Moreover, it was able to increase the activity of the small GTPase, Rac1. These results suggest that the interaction between Tiam1 and MAP1B, is produced by the binding of LC1 with PH domains in Tiam1. The formation of such a complex impacts on the activation levels of Rac1 confirming a novel function of MAP1B related with the control of small GTPases. These results also support the idea of cross-talk between cytoskeleton compartments inside neuronal cells.     

Mutations in Lama1 Disrupt Retinal Vascular Development and Inner Limiting Membrane Formation

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin α1, a basement membrane protein. Despite normal localization of laminin α1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin α1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1^tm1.1Olf, was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2

RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.     

Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser⁶⁹⁶/Thr⁷⁰⁶ and cluster 2, Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser⁸⁶³ phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser⁸⁶³ via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser⁸⁶³ phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser⁸⁶³ as well as on the five other identified sites (e.g. Ser⁸⁵⁹, Ser⁸⁵⁵, Ser⁸⁷⁷, Ser⁶⁹⁶, and Thr⁷⁰⁶). Strikingly, raptor Ser⁸⁶³ phosphorylation is absolutely required for raptor Ser⁸⁵⁹ and Ser⁸⁵⁵ phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser⁸⁶³ phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser⁸⁵⁹ and Ser⁸⁵⁵). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.     

Genetic variability in Puccinia striiformis f. sp. tritici populations sampled on a local scale during natural epidemics

This study investigated genetic polymorphism on a local scale in Puccinia striiformis f. sp. tritici populations during natural epidemics, in four fields located in northern France and sampled in 1998 or 1999. Two hundred and forty-seven isolates were analyzed for their amplified fragment length polymorphism (AFLP) pattern through four primer combinations, and 194 of them were also tested for their virulence factors. Only one or two pathotypes were found in each field, and all isolates had virulence v17, matching the recently introduced Yr17 resistance gene. Polymorphism on a field scale was low. Although 67 loci were polymorphic, 77% of the isolates had the same AFLP pattern, all other patterns being rare or unique. Analyses of the genetic distance between AFLP patterns based on the Jaccard index allowed us to define 12 groups, but a bootstrap analysis showed that all isolates could be assigned to a single clonal lineage. This leads us to conclude that P. striiformis f. sp. tritici populations are clonal on a field scale in northern France.     

Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct

A protocol for the efficient isotopic labeling of large G protein‐coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L‐tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell–cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell–cell communication by the addition of indole during expression. Discrete concentrations of indole and ¹⁵N₂‐L‐tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ～15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681–1690. © 2013 Wiley Periodicals, Inc.