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PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.

Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru β-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis and increased p62 levels. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13Pru-producing cancer types.

Workflows in NGS facilities require high-standard practices and high-throughput pipelines to process the large number of samples received in a timely manner. Downstream protocols such as NGS library preparation require accurate estimation of nucleic acid concentrations, which can be achieved using fluorescent dye-based nucleic acid measurement. Here, we report a protocol for preparing a 384-well Quant-iT PicoGreen assay. The protocol allows the concentrations of 184 DNA samples to be measured simultaneously in duplicate in only 1 h using an Eppendorf epMotion 5075 liquid handling system. The advantages of this high-throughput approach include a reduction in both reagents (10x less reagents compared to a standard protocol) and time (3 h for 384 samples compared with 3 days).

Resident microorganisms are known to influence the fitness and traits of animals under controlled laboratory conditions, but the relevance of these findings to wild animals is uncertain. This study investigated the host functional correlates of microbiota composition in a wild community of three sympatric species of mycophagous drosophilid flies, Drosophila falleni, Drosophila neotestacea and Drosophila putrida. Specifically, we quantified bacterial communities and host transcriptomes by parallel 16S rRNA gene amplicon sequencing and RNA-Seq of individual flies. Among-fly variation in microbiota composition did not partition strongly by sex or species, and included multiple modules, that is, sets of bacterial taxa whose abundance varied in concert across different flies. The abundance of bacteria in several modules varied significantly with multiple host transcripts, especially in females, but the identity of the correlated host transcriptional functions differed with host species, including epithelial barrier function in D. falleni, muscle function in D. putrida, and insect growth and development in D. neotestacea. In D. neotestacea, which harbours the endosymbionts Wolbachia and Spiroplasma, Wolbachia promotes the abundance of Spiroplasma, and is positively correlated with abundance of Lactobacillales and Bacteroidales. Furthermore, most correlations between host gene expression and relative abundance of bacterial modules were co-correlated with abundance of Wolbachia (but not Spiroplasma), indicative of an interdependence between host functional traits, microbiota composition and Wolbachia abundance in this species. These data suggest that, in these natural populations of drosophilid flies, different host species interact with microbial communities in functionally different ways that can vary with the abundance of endosymbionts.

AIM: To assess the genetic structure, biogeography, and the potential for speciation, of a highly host‐specific insect pest with mainland and island populations. LOCATION: East coast of Australia, Lord Howe Island (LHI), and New Zealand. METHODS: We focussed on Mycopsylla fici, a plant sap‐feeding insect host‐specific to the fig tree Ficus macrophylla. We genotyped 152 insects from across the natural and extended host plant range at 14 microsatellite loci and analysed the data using standard population genetics statistics, Discriminant Analysis of Principal Components, genetic autocorrelation, and two Bayesian clustering approaches. RESULTS: Genetic analyses revealed that the northeastern Australian mainland population (Brisbane) is the centre of genetic diversity. Northeastern and southeastern (Sydney) mainland populations are genetically differentiated and interconnected in a stepping‐stone pattern. The LHI population is the most distinct genetically and Bayesian estimates indicated that the most recent colonization occurred c. 2,000‐17,500 years ago from a northeastern mainland origin. In contrast, the New Zealand population is little differentiated from the Sydney population and probably diverged by colonization within the past 200 years. MAIN CONCLUSIONS: The strong differentiation in nuclear microsatellites mirrors previous evidence for divergence of the LHI population from both mtDNA and endosymbiont DNA. The LHI population may be undergoing speciation from the mainland populations, with an oceanic barrier to gene flow. In contrast, the geographically isolated population in Auckland represents a far more recent colonization reflecting the contemporary naturalization of the plant host in New Zealand.

Orthodontic treatment can last between 2 and 3 years. These long treatment times can significantly decrease patient cooperation and yield adverse effects. This review aimed to evaluate the clinical effect of low-level laser therapy (LLLT) on the acceleration of orthodontic tooth movement. We searched the Cochrane Library, PubMed, and EMBASE and included only studies evaluating the effect of LLLT on orthodontic human tooth movement, according to inclusion criteria. We included thirteen studies (three controlled clinical trials [CCTs], five randomized clinical trials [RCTs], and five systematic reviews or meta-analyses). Six of the eight clinical trials showed a statistically significant effect on orthodontic tooth movement toward a low-level laser application. Comparison between studies is difficult because of parameter variability (anchorage, orthodontic appliances, wavelength, and other laser parameters); however, LLLT may be an interesting method to accelerate orthodontic tooth movement. The highest-level studies show that LLLT can accelerate orthodontic movements, but other research must be pursued. This review of the literature seems to suggest concentrating future research regarding the relationship between LLLT and orthodontic tooth movement on wavelengths in infrared radiation approximately 780–810 nm, with a fluence of approximately 5 J/cm2.

The ecology, diversity, and parasitoid complex of plant–sap feeding insects of the family Homotomidae (Hemiptera: Psylloidea) specialised on fig trees (Ficus) have so far received little research attention. They are ecologically important, however, as occasional outbreaks of the homotomid Mycopsylla fici may cause complete defoliation of its host plant, the Moreton Bay fig (Ficus macrophylla). Mycopsylla proxima, the only other species reported from Australia, feeds on F. rubiginosa without any recorded outbreaks. We searched for homotomids and their parasitoids on eight Ficus species on the east coast of Australia, Lord Howe Island (LHI), and in Auckland, New Zealand, and detected them on three Ficus species. Using mitochondrial and nuclear DNA sequences, we delimited three Mycopsylla species, including a putative new species on F. watkinsiana. We also characterised six (including one previously described) parasitoid species of the genus Psyllaephagus (Hymenoptera: Encyrtidae) based on congruent morphological characters and molecular data. Each of the homotomid species was highly host specific to a single fig species, whereas parasitoid species varied in host specificity: three host specific to M. fici and three host generalists. Geographic distribution varied among parasitoid species; e.g. one host-specific species was found on both the mainland and LHI, but a second species only on LHI. Our study revealed previously unrecognised diversity in fig homotomids and especially in their parasitoids. The herbivores and parasitoids showed contrasting patterns of host specificity. Interestingly, M. fici, the only outbreak species, had the highest diversity of associated parasitoid species and was the only species with host-specific parasitoids.

Coevolution between insects and bacterial endosymbionts contributes to the success of many insect lineages. For the first time, we tested for phylogenetic codivergence across multiple taxonomic scales, from within genera to superfamily between 36 psyllid species of seven recognised families (Hemiptera: Psylloidea), their exclusive primary endosymbiont Carsonella and more diverse secondary endosymbionts (S-endosymbionts). Within Aphalaridae, we found that Carsonella and S-endosymbionts were fixed in one Glycaspis and 12 Cardiaspina populations. The dominant S-endosymbiont was Arsenophonus, while Sodalis was detected in one Cardiaspina species. We demonstrated vertical transmission for Carsonella and Arsenophonus in three Cardiaspina species. We found strong support for strict cospeciation and validated the informative content of Carsonella as extended host genome for inference of psyllid relationships. However, S-endosymbiont and host phylogenies were incongruent, and displayed signs of host switching and endosymbiont replacement. The high incidence of Arsenophonus in psyllids and other plant sap-feeding Hemiptera may be due to repeated host switching within this group. In two psyllid lineages, Arsenophonus and Sodalis genes exhibited accelerated evolutionary rates and AT-biases characteristic of long-term host associations. Together with strict vertical transmission and 100% prevalence within host populations, our results suggest an obligate, and not facultative, symbiosis between psyllids and some S-endosymbionts.

Most insects are associated with bacterial symbionts. The bacterial diversity and community composition within hosts may play an important role in shaping insect population biology, ecology and evolution. We focussed on the bacterial microbiome of the Australian fig homotomid Mycopsylla fici (Hemiptera: Psylloidea), which can cause defoliation of its only host tree, Ficus macrophylla. This sap-feeding insect is native to mainland Australia and Lord Howe Island (LHI) but also occurs where its host has been planted, notably in New Zealand. By using a high-throughput 16S rDNA amplicon sequencing approach, we compared the bacterial diversity and community composition in individual adult males of four host populations, Sydney, Brisbane, LHI and Auckland. We also compared males, females and nymphs of the Sydney population. The microbiome of M. fici was simple and consisted mostly of the following three maternally inherited endosymbiont species: the primary endosymbiont Carsonella, a secondary (S-) endosymbiont and Wolbachia. However, the relative abundance of their sequence reads varied between host populations, except for similarities between Sydney and Auckland. In addition, insects from Sydney and Auckland had identical bacterial strains supporting the hypothesis that Sydney is the source population for Auckland. In contrast, mainland and LHI populations harboured the same S-endosymbiont, co-diverged Carsonella but different Wolbachia strains. Besides detecting endosymbiont-specific patterns of either co-evolution or horizontal acquisition, our study highlights that relative abundance of maternally inherited endosymbionts should also be taken into account when studying bacterial communities across host populations, as variations in bacterial density may impact host biology and ecology.

Coevolution between insects and bacterial endosymbionts contributes to the success of many insect lineages. For the first time, we tested for phylogenetic codivergence across multiple taxonomic scales, from within genera to superfamily between 36 psyllid species of seven recognised families (Hemiptera: Psylloidea), their exclusive primary endosymbiont Carsonella and more diverse secondary endosymbionts (S-endosymbionts). Within Aphalaridae, we found that Carsonella and S-endosymbionts were fixed in one Glycaspis and 12 Cardiaspina populations. The dominant S-endosymbiont was Arsenophonus, while Sodalis was detected in one Cardiaspina species. We demonstrated vertical transmission for Carsonella and Arsenophonus in three Cardiaspina species. We found strong support for strict cospeciation and validated the informative content of Carsonella as extended host genome for inference of psyllid relationships. However, S-endosymbiont and host phylogenies were incongruent, and displayed signs of host switching and endosymbiont replacement. The high incidence of Arsenophonus in psyllids and other plant sap-feeding Hemiptera may be due to repeated host switching within this group. In two psyllid lineages, Arsenophonus and Sodalis genes exhibited accelerated evolutionary rates and AT-biases characteristic of long-term host associations. Together with strict vertical transmission and 100% prevalence within host populations, our results suggest an obligate, and not facultative, symbiosis between psyllids and some S-endosymbionts.

While obligate primary (P-) endosymbionts usually cospeciate with their insect hosts, less is known about codiversification of secondary (S-) endosymbionts that are generally considered facultative. Typically, insects of the superfamily Psylloidea harbour one P- ( Carsonella ) and at least one S-endosymbiont, thought to compensate for Carsonella genome reduction. Most codiversification studies have used phylogenies of psyllids and their endosymbionts across and within host families or genera, but few have explored patterns within species. We focussed on P- and S-endosymbionts of three Mycopsylla (Homotomidae) species to explore whether they have congruent phylogenies and within-species geographic structures. The P-endosymbiont Carsonella , a S-endosymbiont and Wolbachia all had 100% prevalence, while Arsenophonus was only found in one species at low prevalence. Congruent phylogenies of Mycopsylla and P-endosymbionts across populations and species support strict cospeciation. S-endosymbiont phylogenies were also congruent across host species but low genetic variation in the S-endosymbiont was not correlated with host phylogeography, possibly due to a shorter evolutionary association. Between species, Wolbachia and Mycopsylla phylogenies were incongruent, probably due to horizontal transmission events. Our study is the first to explore endosymbionts of Mycopsylla and further supports the codivergence of Psylloidea hosts and P-endosymbionts, with obligate host interactions for both P- and S-endosymbionts.

Explanations for the evolution of pathogen-induced fecundity reduction usually rely on a common principle: the trade-off between host longevity and reproduction. Recent advances in nutritional research have, however, challenged this assumption and shown that longevity and reproduction are not inextricably linked. In this study, we showed that beetles infected by cysticercoids of the tapeworm Hymenolepis diminuta increased their total food intake and, more particularly, their carbohydrate consumption compared with uninfected insects. This increased intake was only pronounced during the first 12 days p.i., when the parasite grows and develops into a mature metacestode. Despite consuming more nutrients, infected individuals sustained lower levels of body lipid and were less efficient at converting ingested protein to body protein. However they demonstrated a capacity to compose a diet that sustained high levels of reproductive output unless confined to foods that were nutritionally dilute. We did not find any indication that macronutrient intakes had an effect on host pro-phenoloxidase activity; however, phenoloxidase activity was significantly affected by protein intake. Our results showed that when offered nutritionally complementary diets, infected hosts do not systematically suffer a reduction in fecundity. Thus, in our view, the assumption that a reduction in host reproduction represents an adaptive response by the host or the parasite to divert resources away from reproduction toward other traits should be reassessed.

The Australian fig psylloid, Mycopsylla fici, is a sap-feeding insect herbivore that is host-specific to the Moreton Bay fig, Ficus macrophylla. It has periodic major outbreaks that can cause complete defoliation of individual trees and massive decrease in local leaf and fruit availability, with significant effects for many insect and vertebrate species that utilise the tree’s resources. We used ⅛ of an Illumina MiSeq run to sequence genomic DNA from two pools of five psylloids from two different field sites. We identified 14 polymorphic microsatellite loci and characterised them in 43 individuals from two populations (Sydney and Lord Howe Island, Australia). Within populations, the number of alleles ranged from 4 to 15 per locus with observed heterozygosity of 0–0.9. Four loci deviated from Hardy–Weinberg equilibrium. The microsatellite primers will be useful for the study of population genetics and gene flow within and between psylloid populations.