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Meniere’s disease (MD) is an inner ear disorder characterised by recurrent vertigo attacks associated with sensorineural hearing loss and tinnitus. Evidence from epidemiology and Whole Exome Sequencing (WES) suggests a genetic susceptibility involving multiple genes, including α-Dystrobrevin (DTNA). Here we investigate a Drosophila model. We show that mutation, or knockdown, of the DTNA orthologue in Drosophila, Dystrobrevin (Dyb), results in defective proprioception and impaired function of Johnston’s Organ (JO), the fly’s equivalent of the inner ear. Dyb and another component of the dystrophin-glycoprotein complex (DGC), Dystrophin (Dys), are expressed in support cells within JO. Their specific locations suggest that they form part of support cell contacts, thereby helping to maintain the integrity of the hemolymph-neuron diffusion barrier, which is equivalent to a blood-brain barrier. These results have important implications for the human condition, and notably, we note that DTNA is expressed in equivalent cells of the mammalian inner ear.

The chakra is a cropping system used by the indigenous Kichwa people of the Ecuadorian Amazon that is advocated as a sustainable form of agriculture that ensures food production without impeding ecosystem functioning. Trees and palms are central to the benefits obtained from chakras , however, little is known about the management of these trees and palms. In this research we aimed to understand how the Kichwa people manage these species in chakras and what drives them to do so. We conducted participatory observation research in three Kichwa communities during which we researched 18 chakras belonging to different households. In each chakra we identified the tree and palm species, counted their number, and measured their diameter at 1.3 m aboveground. Additionally, each household was interviewed on the use and management of these species. A total of 740 individual trees and palms were inventoried. Nearly all species in chakras were being actively managed, with the reduction of competition and the protection of seedlings being the most applied practices. This appears to be driven mainly by utilitarian values, as most species were used, most commonly for food and construction. Previous studies indicated that agroecological and mythical values also incite the management of trees and palms, which was not the case in this study. This difference is probably because of cultural erosion or cultural gender roles that affected data acquisition. Three important implications arise from this research: 1) future research should acknowledge and adequately address the large variation that exists among chakras , especially regarding their differences in number and size of trees and palms; 2) trees and palms in chakras should not be assumed to be managed inherently sustainably; 3) the understanding that economic and utilitarian considerations induce active management of palms and trees in chakras can lead to effective conservation policies. • Swidden fallows managed by the Kichwa people (chakras) differ substantially in the number and size of trees and palms. • Trees and palms growing in chakras are managed more intensively than generally assumed. • Economic and utilitarian values, not agroecological or mythical values, drive management of trees and palms in chakras. [ABSTRACT FROM AUTHOR]

Abstract: Live attenuated bacteria are well established as vaccines. Thus, their use as carriers for prophylactic and therapeutic macromolecules is a logical consequence. Here we describe several experimental applications of bacteria to carry heterologous macromolecules into the murine host. First, Listeria monocytogenes are described that are able to transfer eukaryotic expression plasmids into host cells for gene therapy. High multiplicities of infection are still required for efficient gene transfer and we point out some of the bottlenecks that counteract a more efficient transfer and application in vivo. Then, we describe Salmonella enterica serovar Typhimurium (S. typhimurium) as an expression plasmid transfer vehicle for oral DNA vaccination of mice. We demonstrate that the stabilization of the plasmid transformants results in an improved immune response. Stabilization was achieved by replacing the origin of replication of the original high-copy-number plasmid by a low-copy-number origin. Finally, we describe Salmonella carriers for the improved expression of heterologous proteins. We introduce a system in which the plasmid is carried as a single copy during cultivation but is amplified several fold upon infection of the host. Using the same in vivo inducible promoter for both protein expression and plasmid amplification, a substantial increase in antigen expression in vivo can be achieved. A modification of this approach is the introduction of inducible gene expression in vivo with a low-molecular-weight compound. Using P BAD promoter and l-arabinose as inducer we were able to deliberately activate genes in the bacterial carrier. No background activity could be observed with P BAD such that an inducible suicide gene could be introduced. This is adding an important safety feature to such live attenuated carrier bacteria. [Copyright &y& Elsevier]

Axonemal dynein motors are large multi-subunit complexes that drive ciliary movement. Cytoplasmic assembly of these motor complexes involves several co-chaperones, some of which are related to the R2TP co-chaperone complex. Mutations of these genes in humans cause the motile ciliopathy, Primary Ciliary Dyskinesia (PCD), but their different roles are not completely known. Two such dynein (axonemal) assembly factors (DNAAFs) that are thought to function together in an R2TP-like complex are DNAAF4 (DYX1C1) and DNAAF6 (PIH1D3). Here we investigate the Drosophila homologues, CG14921 / Dnaaf4 and CG5048 / Dnaaf6. Surprisingly, Drosophila Dnaaf4 is truncated such that it completely lacks a TPR domain, which in human DNAAF4 is likely required to recruit HSP90. Despite this, we provide evidence that Drosophila Dnaaf4 and Dnaaf6 proteins can associate in an R2TP-like complex that has a conserved role in dynein assembly. Both are specifically expressed and required during the development of the two Drosophila cell types with motile cilia: mechanosensory chordotonal neurons and sperm. Flies that lack Dnaaf4 or Dnaaf6 genes are viable but with impaired chordotonal neuron function and lack motile sperm. We provide molecular evidence that Dnaaf4 and Dnaaf6 are required for assembly of outer dynein arms (ODAs) and a subset of inner dynein arms (IDAs)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lennon, zur Lage, von Kriegsheim and Jarman.)

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans, the impairment of these motors through mutation results in the disease primary ciliary dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila orthologue of the less-known assembly factor DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, The only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The motile cilium/flagellum is an ancient eukaryotic organelle. The molecular machinery of ciliary motility comprises a variety of cilium-specific dynein motor complexes along with other complexes that regulate their activity. Assembling the motors requires the function of dedicated "assembly factors" and transport processes. In humans, mutation of any one of at least 40 different genes encoding components of the motility apparatus causes Primary Ciliary Dyskinesia (PCD), a disease of defective ciliary motility. Recently, Drosophila has emerged as a model for motile cilia biology and motile ciliopathies. This is somewhat surprising as most Drosophila cells lack cilia, and motile cilia are confined to just two specialized cell types: the sperm flagellum with a 9+2 axoneme and the ciliated dendrite of auditory/proprioceptive (chordotonal, Ch) neurons with a 9+0 axoneme. To determine the utility of Drosophila as a model for motile cilia, we survey the Drosophila genome for ciliary motility gene homologs, and assess their expression and function. We find that the molecules of cilium motility are well conserved in Drosophila . Most are readily characterized by their restricted cell-type specific expression patterns and phenotypes. There are also striking differences between the two motile ciliated cell types. Notably, sperm and Ch neuron cilia express and require entirely different outer dynein arm variants-the first time this has been clearly established in any organism. These differences might reflect the specialized functions for motility in the two cilium types. Moreover, the Ch neuron cilia lack the critical two-headed inner arm dynein (I1/f) but surprisingly retain key regulatory proteins previously associated with it. This may have implications for other motile 9+0 cilia, including vertebrate embryonic nodal cilia required for left-right axis asymmetry. We discuss the possibility that cell-type specificity in ciliary motility machinery might occur in humans, and therefore underlie some of the phenotypic variation observed in PCD caused by different gene mutations. Our work lays the foundation for the increasing use of Drosophila as an excellent model for new motile ciliary gene discovery and validation, for understanding motile cilium function and assembly, as well as understanding the nature of genetic defects underlying human motile ciliopathies.

The massive dynein motor complexes that drive ciliary and flagellar motility require cytoplasmic preassembly, a process requiring dedicated dynein assembly factors (DNAAFs). How DNAAFs interact with molecular chaperones to control dynein assembly is not clear. By analogy with the well-known multifunctional HSP90-associated cochaperone, R2TP, several DNAAFs have been suggested to perform novel R2TP-like functions. However, the involvement of R2TP itself (canonical R2TP) in dynein assembly remains unclear. Here we show that in Drosophila melanogaster , the R2TP-associated factor, Wdr92, is required exclusively for axonemal dynein assembly, likely in association with canonical R2TP. Proteomic analyses suggest that in addition to being a regulator of R2TP chaperoning activity, Wdr92 works with the DNAAF Spag1 at a distinct stage in dynein preassembly. Wdr92/R2TP function is likely distinct from that of the DNAAFs proposed to form dynein-specific R2TP-like complexes. Our findings thus establish a connection between dynein assembly and a core multifunctional cochaperone., (© 2018 zur Lage et al.)

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of 'client' proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease., Competing Interests: GM, PY, SM, DD, PT, MK, Pz, AS, AG, AS, HT, FE, ST, Av, AJ, PM No competing interests declared, (© 2018, Mali et al.)

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.

In a variety of organisms, early neurogenesis requires the function of basic-helix-loop-helix (bHLH) transcription factors. For the Drosophila PNS, such transcription factors are encoded by the proneural genes (atonal and the achaete-scute complex, AS-C). We have identified a proneural gene, amos, that has strong similarity with atonal in its bHLH domain. We present evidence that amos is required for olfactory sensilla and is regulated by the prepattern gene lozenge. Between them, amos, atonal, and the AS-C can potentially account for the origin of the entire PNS.

A semi-lethal, sterile allele of the smooth locus (2-91.5), sm3, was discovered in an artificial selection line for low abdominal bristle number that had been started from a P-M dysgenic cross. The fitness effects and extremely low bristle number phenotype of the allele could not be separated by recombination from a P-element insertion at cytological location 56E, and precise excision of the P-element at this site was associated with reversion to wild type. The smooth gene was cloned using the P-element insertion as a tag. The gene encodes a 2.6-kb transcript derived from 10 exons and covers a genomic region of at least 80 kb. The Drosophila smooth gene shares substantial sequence identity with a group of RNA binding proteins, with the closest relationship being to the human heterogeneous nuclear ribonucleoprotein L gene. The smooth gene is by definition an abdominal bristle number quantitative trait locus, but further work is required to discern whether naturally occurring allelic variation at this locus is a source of genetic variation for abdominal bristle number in natural populations.