DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10³ CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.     

Genes controlling legume nodule numbers affect phenotypic plasticity responses to nitrogen in the presence and absence of rhizobia

We investigated the role of three autoregulation of nodulation (AON) genes in regulating of root and shoot phenotypes when responding to changing nitrogen availability in the model legume, Medicago truncatula. These genes, RDN1‐1 (ROOT DETERMINED NODULATION1‐1), SUNN (SUPER NUMERIC NODULES), and LSS (LIKE SUNN SUPERNODULAOR), act in a systemic signalling pathway that limits nodule numbers. This pathway is also influenced by nitrogen availability, but it is not well known if AON genes control root and shoot phenotypes other than nodule numbers in response to nitrogen. We conducted a controlled glasshouse experiment to compare root and shoot phenotypes of mutants and wild type plants treated with four nitrate concentrations. All AON mutants showed altered rhizobia‐independent phenotypes, including biomass allocation, lateral root length, lateral root density, and root length ratio. In response to nitrogen, uninoculated AON mutants were less plastic than the wild type in controlling root mass ratio, root length ratio, and lateral root length. This suggests that AON genes control nodulation‐independent root architecture phenotypes in response to nitrogen. The phenotypic differences between wild type and AON mutants were exacerbated by the presence of nodules, pointing to resource competition as an additional mechanism affecting root and shoot responses to nitrogen.     

Terrestrial mammal surveillance using hybridization capture of environmental DNA from African waterholes

Determining species distributions can be extremely challenging but is crucial to ecological and conservation research. Environmental DNA (eDNA) approaches have shown particular promise in aquatic systems for several vertebrate and invertebrate species. For terrestrial animals, however, eDNA‐based surveys are considerably more difficult due to the lack of or difficulty in obtaining appropriate sampling substrate. In water‐limited ecosystem where terrestrial mammals are often forced to congregate at waterholes, water and sediment from shared water sources may be a suitable substrate for noninvasive eDNA approaches. We characterized mitochondrial DNA sequences from a broad range of terrestrial mammal species in two different African ecosystems (in Namibia and Tanzania) using eDNA isolated from native water, sediment and water filtered through glass fibre filters. A hybridization capture enrichment with RNA probes targeting the mitochondrial genomes of 38 mammal species representing the genera/families expected at the respective ecosystems was employed, and 16 species were identified, with a maximum mitogenome coverage of 99.8%. Conventional genus‐specific PCRs were tested on environmental samples for two genera producing fewer positive results than hybridization capture enrichment. An experiment with mock samples using DNA from non‐African mammals showed that baits covering 30% of nontarget mitogenomes produced 91% mitogenome coverage after capture. In the mock samples, over‐representation of DNA of one species still allowed for the detection of DNA of other species that was at a 100‐fold lower concentration. Hybridization capture enrichment of eDNA is therefore an effective method for monitoring terrestrial mammal species from shared water sources.     

Heat Stability of Differently Stabilized Solid Lipid Nanoparticles in the Presence of Excess Bulk Phase Protein

In order to apply emulsion-based delivery systems to food, they have to be stable in a protein rich environment. This study investigated the stability of solid lipid nanoparticles (SLN) during heat treatment in the presence or absence of β-lactoglobulin (BLG). SLN were stabilized either by Tween 20 (TS) or by the protein itself (BS) and were enriched to a total BLG content of 56 mg/mL. The sizes of both types of SLN were initially in the range of 170 nm. The amount of free protein was determined before and after enrichment with BLG. As revealed by particle size and zeta potential measurements, a protein layer of BLG (hard corona) adsorbed on BS but not on TS. By contrast, a soft corona was formed around both BS and TS. SLN were heat treated in the presence and absence of protein and were characterized regarding size and zeta potential. According to transmission electron microscopy imaging, heating did not affect the shape of TS and BS: TS were platelets, whereas BS exhibited a spherical or platelet like shape. Upon heat treatment, the particle size of TS increased to about 3.5 fold of the initial size (to appr. 600 nm) in the presence and in the absence of excess protein. The cloudy protein layer (soft corona) around TS could thus not prevent coalescence of TS. By contrast, BS did not experience a change in particle size. Hence, by the choice of emulsifier, an encapsulation system that is stable against heat treatment can be obtained.

     

Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale

Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species.     

Reversible Membrane Pearling in Live Cells upon Destruction of the Actin?Cortex

Membrane pearling in live cells is observed when the plasma membrane is depleted of its support, the cortical actin network. Upon efficient depolymerization of actin, pearls of variable size are formed, which are connected by nanotubes of ∼40 nm diameter. We show that formation of the membrane tubes and their transition into chains of pearls do not require external tension, and that they neither depend on microtubule-based molecular motors nor pressure generated by myosin-II. Pearling thus differs from blebbing. The pearling state is stable as long as actin is prevented from polymerizing. When polymerization is restored, the pearls are retracted into the cell, indicating continuity of the membrane. Our data suggest that the alternation of pearls and strings is an energetically favored state of the unsupported plasma membrane, and that one of the functions of the actin cortex is to prevent the membrane from spontaneously assuming this configuration.     

Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging

Nonstructural protein 5A (NS5A) of bovine viral diarrhea virus (BVDV) is a hydrophilic phosphoprotein with RNA binding activity and a critical component of the viral replicase. In silico analysis suggests that NS5A encompasses three domains interconnected by two low-complexity sequences (LCSs). While domain I harbors two functional determinants, an N-terminal amphipathic helix important for membrane association, and a Zn-binding site essential for RNA replication, the structure and function of the C-terminal half of NS5A are still ill defined. In this study, we introduced a panel of 10 amino acid deletions covering the C-terminal half of NS5A. In the context of a highly efficient monocistronic replicon, deletions in LCS I and the N-terminal part of domain II, as well as in domain III, were tolerated with regard to RNA replication. When introduced into a bicistronic replicon, only deletions in LCS I and the N-terminal part of domain II were tolerated. In the context of the viral full-length genome, these mutations allowed residual virion morphogenesis. Based on these data, a functional monocistronic BVDV replicon coding for an NS5A variant with an insertion of the fluorescent protein mCherry was constructed. Live cell imaging demonstrated that a fraction of NS5A-mCherry localizes to the surface of lipid droplets. Taken together, this study provides novel insights into the functions of BVDV NS5A. Moreover, we established the first pestiviral replicon expressing fluorescent NS5A-mCherry to directly visualize functional viral replication complexes by live cell imaging.     

Polyubiquitinated Tristetraprolin Protects from TNF-induced,Caspase-mediated Apoptosis

Binding of TNF to its receptor (TNFR1) elicits the spatiotemporal assembly of two signaling complexes that coordinate the balance between cell survival and cell death. We have shown previously that, following TNF treatment, the mRNA decay protein tristetraprolin (TTP) is Lys-63-polyubiquitinated by TNF receptor-associated factor 2 (TRAF2), suggesting a regulatory role in TNFR signaling. Here we demonstrate that TTP interacts with TNFR1 in a TRAF2-dependent manner, thereby initiating the MEKK1/MKK4-dependent activation of JNK activities. This regulatory function toward JNK activation but not NF-κB activation depends on lysine 105 of TTP, which we identified as the corresponding TRAF2 ubiquitination site. Disabling TTP polyubiquitination results in enhanced TNF-induced apoptosis in cervical cancer cells. Together, we uncover a novel aspect of TNFR1 signaling where TTP, in alliance with TRAF2, acts as a balancer of JNK-mediated cell survival versus death.     

Cryptic diversity within the rotifer Polyarthra dolichoptera along an altitudinal gradient

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