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β-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbb β-thalassemia mouse and hemoglobin E (HbE)/β-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbb mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in β-thalassemia patients. In Hbb mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbb neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GLy6C) appeared as band form cells, and a striking expansion of immature (Ly6GLy6C) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbb neutrophils. In addition, in vivo infection with failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbb mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/β-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in β-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.

BACKGROUND Insecticide targets are often identified by mutations that confer resistance, but the intricacies of insecticide binding and downstream processes leading to insect death often remain obscure. Mutations in α6‐like nicotinic acetylcholine receptor subunit genes have been associated with high levels of resistance to spinosad in many insect species, including Drosophila melanogaster. Here, we aimed to expand our understanding of the effects of the natural product insecticide spinosad on its protein target, the α6 subunit, using genetic tools available in D. melanogaster. RESULTS Functional, fluorescently tagged Dα6 subunits (Dα6YFP) were developed to allow observation of the protein in vivo. Larvae expressing Dα6YFP were exposed to a sub‐lethal concentration of spinosyn A (0.025 ppm) for 6 days, leading to a 64% reduction in fluorescence relative to unexposed larvae. Direct application of high doses of spinosyn A to dissected larval brains resulted in a visible 38.25% decrease in Dα6YFP within 20 min, indicating that degradation of the Dα6 protein occurred in response to spinosyn A exposure. Chemical inhibition of the proteasome system using the multiple myeloma treatment drug, PS‐341 reduced loss of Dα6YFP in response to spinosyn A at the 20‐min time point to 6.35%. In addition, in vivo administration of PS‐341 prior to spinosad exposure reduced the effect of spinosad on larval activity. CONCLUSION Based on these data, we propose that exposure to spinosad leads to degradation of the α6‐like target protein, a potentially novel element in the mode of action of spinosyns that may contribute to their toxicity towards insects. © 2021 Society of Chemical Industry

Background Larvae of the Australian sheep blowfly, Lucilia cuprina, parasitise sheep by feeding on skin excretions, dermal tissue and blood, causing severe damage known as flystrike or myiasis. Recent advances in -omic technologies and bioinformatic data analyses have led to a greater understanding of blowfly biology and should allow the identification of protein families involved in host-parasite interactions and disease. Current literature suggests that proteins of the SCP (Sperm-Coating Protein)/TAPS (Tpx-1/Ag5/PR-1/Sc7) (SCP/TAPS) superfamily play key roles in immune modulation, cross-talk between parasite and host as well as developmental and reproductive processes in parasites. Methods Here, we employed a bioinformatics workflow to curate the SCP/TAPS protein gene family in L. cuprina. Protein sequence, the presence and number of conserved CAP-domains and phylogeny were used to group identified SCP/TAPS proteins; these were compared to those found in Drosophila melanogaster to make functional predictions. In addition, transcription levels of SCP/TAPS protein-encoding genes were explored in different developmental stages. Results A total of 27 genes were identified as belonging to the SCP/TAPS gene family: encoding 26 single-domain proteins each with a single CAP domain and a solitary double-domain protein containing two conserved cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domains. Surprisingly, 16 SCP/TAPS predicted proteins formed an extended tandem array spanning a 53 kb region of one genomic region, which was confirmed by MinION long-read sequencing. RNA-seq data indicated that these 16 genes are highly transcribed in all developmental stages (excluding the embryo). Conclusions Future work should assess the potential of selected SCP/TAPS proteins as novel targets for the control of L. cuprina and related parasitic flies of major socioeconomic importance.