Hedgehog signaling in the Drosophila eye and head: an analysis of the effects of different patched trans-heterozygotes

Characterization of different alleles of the Hedgehog receptor patched (ptc) indicates that they can be grouped into several classes. Most mutations result in complete loss of Ptc function. However, missense mutations located within the putative sterol-sensing domain (SSD) or C terminus of ptc encode antimorphic proteins that are unable to repress Smo activity and inhibit wild-type Ptc from doing so, but retain the ability to bind and sequester Hh. Analysis of the eye and head phenotypes of Drosophila melanogaster in various ptc/ptc(tuf1) heteroallelic combinations shows that these two classes of ptc allele can be easily distinguished by their eye phenotype, but not by their head phenotype. Adult eye size is inversely correlated with head vertex size, suggesting an alteration of cell fate within the eye-antennal disc. A balance between excess cell division and cell death in the mutant eye discs may also contribute to final eye size. In addition, contrary to results reported recently, the role of Hh signaling in the Drosophila head vertex appears to be primarily in patterning rather than in proliferation, with Ptc and Smo having opposing effects on formation of medial structures.     

Manipulation of the seagrass-associated microbiome reduces disease severity

Host-associated microbes influence host health and function and can be a first line of defence against infections. While research increasingly shows that terrestrial plant microbiomes contribute to bacterial, fungal, and oomycete disease resistance, no comparable experimental work has investigated marine plant microbiomes or more diverse disease agents. We test the hypothesis that the eelgrass (Zostera marina) leaf microbiome increases resistance to seagrass wasting disease. From field eelgrass with paired diseased and asymptomatic tissue, 16S rRNA gene amplicon sequencing revealed that bacterial composition and richness varied markedly between diseased and asymptomatic tissue in one of the two years. This suggests that the influence of disease on eelgrass microbial communities may vary with environmental conditions. We next experimentally reduced the eelgrass microbiome with antibiotics and bleach, then inoculated plants with Labyrinthula zosterae, the causative agent of wasting disease. We detected significantly higher disease severity in eelgrass with a native microbiome than an experimentally reduced microbiome. Our results over multiple experiments do not support a protective role of the eelgrass microbiome against L. zosterae. Further studies of these marine host–microbe–pathogen relationships may continue to show new relationships between plant microbiomes and diseases.     

Identification and characterization of a novel amphioxus dopamine D1-like receptor

Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D₁ and D₂) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus ( Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D₁-like receptor sequences. One of these receptors, AmphiD₁/β, was found to have high levels of sequence similarity to both vertebrate D₁ receptors and to β-adrenergic receptors. Here, we report on the cloning of AmphiD₁/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD₁/β has a similar pharmacology to vertebrate D₁ receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits 'agonist-specific coupling' depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD₁/β may be an amphioxus orthologue of vertebrate β-adrenergic receptors.     

Diversity of dimethylsulfide-degrading methanogens and sulfate-reducing bacteria in anoxic sediments along the Medway Estuary,UK

Methane is a powerful greenhouse gas but the microbial diversity mediating methylotrophic methanogenesis is not well-characterized. One overlooked route to methane is via the degradation of dimethylsulfide (DMS), an abundant organosulfur compound in the environment. Methanogens and sulfate-reducing bacteria (SRB) can degrade DMS in anoxic sediments depending on sulfate availability. However, we know little about the underlying microbial community and how sulfate availability affects DMS degradation in anoxic sediments. We studied DMS-dependent methane production along the salinity gradient of the Medway Estuary (UK) and characterized, for the first time, the DMS-degrading methanogens and SRB using cultivation-independent tools. DMS metabolism resulted in high methane yield (39%–42% of the theoretical methane yield) in anoxic sediments regardless of their sulfate content. Methanomethylovorans, Methanolobus and Methanococcoides were dominant methanogens in freshwater, brackish and marine incubations respectively, suggesting niche-partitioning of the methanogens likely driven by DMS amendment and sulfate concentrations. Adding DMS also led to significant changes in SRB composition and abundance in the sediments. Increases in the abundance of Sulfurimonas and SRB suggest cryptic sulfur cycling coupled to DMS degradation. Our study highlights a potentially important pathway to methane production in sediments with contrasting sulfate content and sheds light on the diversity of DMS degraders.     

Investigating Models of Protein Function and Allostery With a Widespread Mutational Analysis of a Light-Activated Protein

To investigate the relationship between a protein’s sequence and its biophysical properties, we studied the effects of more than 100 mutations in Avena sativa light-oxygen-voltage domain 2, a model protein of the Per-Arnt-Sim family. The A. sativa light–oxygen–voltage domain 2 undergoes a photocycle with a conformational change involving the unfolding of the terminal helices. Whereas selection studies typically search for winners in a large population and fail to characterize many sites, we characterized the biophysical consequences of mutations throughout the protein using NMR, circular dichroism, and ultraviolet/visible spectroscopy. Despite our intention to introduce highly disruptive substitutions, most had modest or no effect on function, and many could even be considered to be more photoactive. Substitutions at evolutionarily conserved sites can have minimal effect, whereas those at nonconserved positions can have large effects, contrary to the view that the effects of mutations, especially at conserved positions, are predictable. Using predictive models, we found that the effects of mutations on biophysical function and allostery reflect a complex mixture of multiple characteristics including location, character, electrostatics, and chemistry.     

Adolescent Changes in Dopamine D1 Receptor Expression in Orbitofrontal Cortex and Piriform Cortex Accompany an Associative Learning Deficit

The orbitofrontal cortex (OFC) and piriform cortex are involved in encoding the predictive value of olfactory stimuli in rats, and neural responses to olfactory stimuli in these areas change as associations are learned. This experience-dependent plasticity mirrors task-related changes previously observed in mesocortical dopamine neurons, which have been implicated in learning the predictive value of cues. Although forms of associative learning can be found at all ages, cortical dopamine projections do not mature until after postnatal day 35 in the rat. We hypothesized that these changes in dopamine circuitry during the juvenile and adolescent periods would result in age-dependent differences in learning the predictive value of environmental cues. Using an odor-guided associative learning task, we found that adolescent rats learn the association between an odor and a palatable reward significantly more slowly than either juvenile or adult rats. Further, adolescent rats displayed greater distractibility during the task than either juvenile or adult rats. Using real-time quantitative PCR and immunohistochemical methods, we observed that the behavioral deficit in adolescence coincides with a significant increase in D1 dopamine receptor expression compared to juvenile rats in both the OFC and piriform cortex. Further, we found that both the slower learning and increased distractibility exhibited in adolescence could be alleviated by experience with the association task as a juvenile, or by an acute administration of a low dose of either the dopamine D1 receptor agonist SKF-38393 or the D2 receptor antagonist eticlopride. These results suggest that dopaminergic modulation of cortical function may be important for learning the predictive value of environmental stimuli, and that developmental changes in cortical dopaminergic circuitry may underlie age-related differences in associative learning.     

Late‐onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5^S163R/wt), and homozygous knock‐in (Ctrp5^S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5^S163R/S163R and Ctrp5^S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.     

Targeting human CALR‐mutated MPN progenitors with a neoepitope‐directed monoclonal antibody

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co‐expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK‐STAT signalling, TPO‐independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR‐dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.