The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.

We report the first discovery since the 1970s of a new extant family (Aenigmatineidae fam.n.) of homoneurous moths, based on the small Aenigmatinea glatzella sp.n. from Kangaroo Island off southern Australia. It exhibits a combination of extraordinary anatomical characters, and, unlike most homoneurous moths, its larva is a conifer‐feeder (stem mining in Callitris, Cupressaceae). While the adult's mouthparts are strongly regressed, evidence from other morphological characters and from a Bayesian analysis of 25 genetic loci convincingly places the taxon among Glossata (‘tongue moths’). An unexpected tongue moth clade including Acanthopteroctetidae and Neopseustidae, suggested with low support in recent molecular analyses, remarkably becomes strongly supported when Aenigmatinea is included in the molecular analysis; the new taxon becomes subordinated in that clade (as sister group to Neopseustidae) and the clade itself appears as the sister group of all Heteroneura, representing the vast majority of all Lepidoptera. Including Aenigmatinea into the analysis thereby strengthens the surprising indication of non‐monophyly of Myoglossata, and the new phylogeny requires an additional number of ad hoc assumptions of convergence/character reversals in early Lepidoptera evolution. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:44393B52-1889-431A-AB08-6BBCF8F946B8.

Heliozelidae are a widespread, evolutionarily early diverging family of small, day-flying monotrysian moths, for which a comprehensive phylogeny is lacking. We generated the first molecular phylogeny of the family using DNA sequences of two mitochondrial genes (COI and COII) and two nuclear genes (H3 and 28S) from 130 Heliozelidae specimens, including eight of the twelve known genera: Antispila, Antispilina, Coptodisca, Heliozela, Holocacista, Hoplophanes, Pseliastis, and Tyriozela. Our results provide strong support for five major Heliozelidae clades: (i) a large widespread clade containing the leaf-mining genera Antispilina, Coptodisca and Holocacista and some species of Antispila, (ii) a clade containing most of the described Antispila, (iii) a clade containing the leaf-mining genus Heliozela and the monotypic genus Tyriozela, (iv) an Australian clade containing Pseliastis and (v) an Australian clade containing Hoplophanes. Each clade includes several new species and potentially new genera. Collectively, our data uncover a rich and undescribed diversity that appears to be especially prevalent in Australia. Our work highlights the need for a major taxonomic revision of the family and for generating a robust molecular phylogeny using multi-gene approaches in order to resolve the relationships among clades.

Repair after damage is essential for tissue homeostasis. Postmenstrual endometrial repair is a cyclical manifestation of rapid, scar-free, tissue repair taking ∼3-5 d. Skin repair after wounding is slower (∼2 wk). In the case of chronic wounds, it takes months to years to restore integrity. Herein, the unique "rapid-repair" endometrial environment is translated to the "slower repair" skin environment. Menstrual fluid (MF), the milieu of postmenstrual endometrial repair, facilitates healing of endometrial and keratinocyte "wounds" in vitro, promoting cellular adhesion and migration, stimulates keratinocyte migration in an ex vivo human skin reconstruct model, and promotes re-epithelialization in an in vivo porcine wound model. Proteomic analysis of MF identified a large number of proteins: migration inhibitory factor, neutrophil gelatinase-associated lipocalin, follistatin like-1, chemokine ligand-20, and secretory leukocyte protease inhibitor were selected for further investigation. Functionally, they promote repair of endometrial and keratinocyte wounds by promoting migration. Translation of these and other MF factors into a migration-inducing treatment paradigm could provide novel treatments for tissue repair.-Evans, J., Infusini, G., McGovern, J., Cuttle, L., Webb, A., Nebl, T., Milla, L., Kimble, R., Kempf, M., Andrews, C. J., Leavesley, D., Salamonsen, L. A. Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair.

Heliozelidae are a cosmopolitan family of small, day-flying moths, and include some pest species of commercial crops. Overall, the family is poorly known and lacks a well-resolved phylogeny. Previous molecular and taxonomic work has revealed rich undescribed diversity within the family, particularly in Australia; however, the relationships amongst the major clades or genera were not resolved. We sequenced the transcriptomes of 39 taxa, representing all major genera of Heliozelidae, and seven outgroups representing most other Adeloidea families and the putative sister superfamily, Andesianoidea. The resulting phylogeny, based on the coding sequences of up to 1049 nuclear genes, provides a robust hypothesis for the generic relationships within Heliozelidae. On the basis of this analysis, the genus Plesiozela, previously proposed sister group of all other Heliozelidae, is excluded from the family and formally transferred to Incurvariidae. By incorporating fossil and secondary time calibrations into our phylogeny, we estimated that Heliozelidae ancestors first appeared at the beginning of the Late Cretaceous, approximately 95 Mya. We propose an ancestral biogeographical range hypothesis of the family, based on a combination of our transcriptome data and a previous multigene study including over 100 species. Our ancestral range modelling results suggest Heliozelidae are likely to have originated in the Australian region, with subsequent range expansions to the rest of the world.

Alpine plant–pollinator communities play an important role in the functioning of alpine ecosystems, which are highly threatened by climate change. However, we still have a poor understanding of how environmental factors and spatiotemporal variability shape these communities. Here, we investigate what drives structure and beta diversity in a plant–pollinator metacommunity from the Australian alpine region using two approaches: pollen DNA metabarcoding (MB) and observations. Individual pollinators often carry pollen from multiple plant species, and therefore we expected MB to reveal a more diverse and complex network structure. We used two gene regions (ITS2 and trnL) to identify plant species present in the pollen loads of 154 insect pollinator specimens from three alpine habitats and construct MB networks, and compared them to networks based on observations alone. We compared species and interaction turnover across space for both types of networks, and evaluated their differences for plant phylogenetic diversity and beta diversity. We found significant structural differences between the two types of networks; notably, MB networks were much less specialized but more diverse than observation networks, with MB detecting many cryptic plant species. Both approaches revealed that alpine pollination networks are very generalized, but we estimated a high spatial turnover of plant species (0.79) and interaction rewiring (0.6) as well as high plant phylogenetic diversity (0.68) driven by habitat differences based on the larger diversity of plant species and species interactions detected with MB. Overall, our findings show that habitat and microclimatic heterogeneity drives diversity and fine‐scale spatial turnover of alpine plant–pollinator networks.

Monitoring biodiversity is a growing and pressing challenge, particularly as climate change threatens species with extinction and leads to widespread shifts in plant distribution and phenology. Tracking changes via ground vegetation surveys is costly and time‐consuming, hence monitoring of complex and heterogenous communities remains an ongoing challenge. Molecular DNA methods are rapidly being developed to provide fast and reproducible results for environmental monitoring, including diet and ecosystem assessments. Here, we used DNA metabarcoding of pollen foraged by European honey bees (Apis mellifera) to investigate their floral resource use in an urban reserve. We collected three different pollen samples from hives: individual bees, raw honey and pollen traps, and identified plants using two metabarcoding markers (ITS2 and trnL). We then compared the results to a ground vegetation survey of surrounding flowering taxa. Pollen DNA metabarcoding detected 74 taxa (48.6% identified to species) across all pollen sources, compared to 44 taxa recorded by the survey (93% identified to species). Within the metabarcoding results, we identified 25% of the genera and 9% of the species found during the survey, with three of the top 10 flowering genera represented. While honey was the most taxon‐rich pollen source (mean = 8.5, SD = 3.5), followed by honey bees (mean = 5.8, SD = 6.1) and pollen traps (mean = 4.2, SD = 1.7), combining the results of six individual bees could detect similar taxa numbers to honey, while 20 bees were required to detect as many taxa as the survey. We demonstrate how DNA metabarcoding of the pollen foraged by honey bees can detect more flowering taxa than traditional survey methods, and how different pollen sources and genetic markers affect the level of detection of plant taxa. The foraging choices of honey bees matched few species detected by the vegetation survey, therefore pollen metabarcoding is recommended as a complementary approach to ground surveys. Rigorous validation and stringent filtering of metabarcoding results were also required to exclude potential false positives. Altogether, this molecular approach can be used to augment vegetation surveys, while tracking the floral resources used by bees.