Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3’s ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3’s ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.     

Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus)

More than 68 billion chickens were produced globally in 2018, emphasising their major contribution to the production of protein for human consumption and the importance of their pathogens. Protozoan Eimeria spp. are the most economically significant parasites of chickens, incurring global costs of more than UK £10.4 billion per annum. Seven Eimeria spp. have long been recognised to infect chickens, with three additional cryptic operational taxonomic units (OTUs) first described more than 10 years ago. As the world’s farmers attempt to reduce reliance on routine use of antimicrobials in livestock production, replacing drugs that target a wide range of microbes with precise species- and sometimes strain-specific vaccines, the breakthrough of cryptic genetic types can pose serious problems. Consideration of biological characteristics including oocyst morphology, pathology caused during infection and pre-patent periods, combined with gene-coding sequences predicted from draft genome sequence assemblies, suggest that all three of these cryptic Eimeria OTUs possess sufficient genetic and biological diversity to be considered as new and distinct species. The ability of these OTUs to compromise chicken bodyweight gain and escape immunity induced by current commercially available anticoccidial vaccines indicates that they could pose a notable threat to chicken health, welfare, and productivity. We suggest the names Eimeria lata n. sp., Eimeria nagambie n. sp. and Eimeria zaria n. sp. for OTUs x, y and z, respectively, reflecting their appearance (x) or the origins of the first isolates of these novel species (y, z).     

Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR

The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.     

Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam,with marked variability by land use type

Azole-resistant environmental Aspergillus fumigatus presents a threat to public health but the extent of this threat in Southeast Asia is poorly described. We conducted environmental surveillance in the Mekong Delta region of Vietnam, collecting air and ground samples across key land-use types, and determined antifungal susceptibilities of Aspergillus section Fumigati (ASF) isolates and azole concentrations in soils. Of 119 ASF isolates, 55% were resistant (or non-wild type) to itraconazole, 65% to posaconazole and 50% to voriconazole. Azole resistance was more frequent in A. fumigatus sensu stricto isolates (95%) than other ASF species (32%). Resistant isolates and agricultural azole residues were overrepresented in samples from cultivated land. cyp51A gene sequence analysis showed 38/56 resistant A. fumigatus sensu stricto isolates carried known resistance mutations, with TR₃₄/L98H most frequent (34/38).     

What triggers the “bail out” behaviour in the limpet Lottia gigantea?

Manipulating objects (small rocks, mussel shells, etc.) to which limpets are clinging will cause many spontaneously to release their hold ("bail out"). Experiments demonstrated that in the owl limpet (Lottia gigantea) bail out was only triggered by rotation of the limpet around a horizontal axis at rates of 0.13 to 1.00rps. Limpets reattached more vigorously to a surface following a “bail out” than when they were forcibly removed from the substratum. In the field a variety of intertidal gastropods were observed to “bail out”. “Bail out” appears to be an adaptive response allowing an individual to abandon a dislodged substratum for a more stable one.     

Detrital diversity influences estuarine ecosystem performance

Global losses of seagrasses and mangroves, eutrophication‐driven increases in ephemeral algae, and macrophyte invasions have impacted estuarine detrital resources. To understand the implications of these changes on benthic ecosystem processes, we tested the hypotheses that detrital source richness, mix identity, and biomass influence benthic primary production, metabolism, and nutrient fluxes. On an estuarine muddy sandflat, we manipulated the availability of eight detrital sources, including mangrove, seagrass, and invasive and native algal species that have undergone substantial changes in distribution. Mixes of these detrital sources were randomly assigned to one of 12 treatments and dried detrital material was added to seventy‐two 0.25 m² plots (n = 6 plots). The treatments included combinations of either two or four detrital sources and high (60 g) or low (40 g) levels of enrichments. After 2 months, the dark, light, and net uptake of NH₄⁺, dissolved inorganic nitrogen, and the dark efflux of dissolved organic nitrogen were each significantly influenced by the identity of detrital mixes, rather than detrital source richness or biomass. However, gross and net primary productivity, average oxygen flux, and net NO_X and dissolved inorganic phosphorous fluxes were significantly greater in treatments with low than with high detrital source richness. These results demonstrate that changes in detrital source richness and mix identity may be important drivers of estuarine ecosystem performance. Continued impacts to estuarine macrophytes may, therefore, further alter detritus‐fueled productivity and processes in estuaries. Specific tests that address predicted future changes to detrital resources are required to determine the consequences of this significant environmental problem.     

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD

Background

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids.

Methods

Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry.

Results

There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10^-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all).

Conclusions

Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.