Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform.

The majority of clinically approved drugs target proteins that are secreted or cell surface bound. However, further advances in this area have been hindered by the challenging nature of receptor deorphanization, as there are still many secreted and cell-bound proteins with unknown binding partners. Here, we developed an advanced screening platform that combines CRISPR-CAS9 guide-mediated gene activation (CRISPRa) and high-avidity bead-based selection. The CRISPRa platform incorporates serial enrichment and flow cytometry-based monitoring, resulting in substantially improved screening sensitivity for well-known yet weak interactions of the checkpoint inhibitor family. Our approach has successfully revealed that siglec-4 exerts regulatory control over T cell activation through a low affinity trans-interaction with the costimulatory receptor 4-1BB. Our highly efficient screening platform holds great promise for identifying extracellular interactions of uncharacterized receptor-ligand partners, which is essential to develop next-generation therapeutics, including additional immune checkpoint inhibitors.

Route of drug administration in out-of-hospital cardiac arrest: A protocol for a randomised controlled trial (PARAMEDIC-3).

Aims: The PARAMEDIC-3 trial evaluates the clinical and cost-effectiveness of an intraosseous first strategy, compared with an intravenous first strategy, for drug administration in adults who have sustained an out-of-hospital cardiac arrest. Methods: PARAMEDIC-3 is a pragmatic, allocation concealed, open-label, multi-centre, superiority randomised controlled trial. It will recruit 15,000 patients across English and Welsh ambulance services. Adults who have sustained an out-of-hospital cardiac arrest are individually randomised to an intraosseous access first strategy or intravenous access first strategy in a 1:1 ratio through an opaque, sealed envelope system. The randomised allocation determines the route used for the first two attempts at vascular access. Participants are initially enrolled under a deferred consent model.The primary clinical-effectiveness outcome is survival at 30-days. Secondary outcomes include return of spontaneous circulation, neurological functional outcome, and health-related quality of life. Participants are followed-up to six-months following cardiac arrest. The primary health economic outcome is incremental cost per quality-adjusted life year gained. Conclusion: The PARAMEDIC-3 trial will provide key information on the clinical and cost-effectiveness of drug route in out-of-hospital cardiac arrest.Trial registration: ISRCTN14223494, registered 16/08/2021, prospectively registered.

HPV+ head and neck cancer-derived small extracellular vesicles communicate with TRPV1+ neurons to mediate cancer pain.

ABSTRACT: Severe pain is often experienced by patients with head and neck cancer and is associated with a poor prognosis. Despite its frequency and severity, current treatments fail to adequately control cancer-associated pain because of our lack of mechanistic understanding. Although recent works have shed some light of the biology underlying pain in HPV-negative oral cancers, the mechanisms mediating pain in HPV+ cancers remain unknown. Cancer-derived small extracellular vesicles (cancer-sEVs) are well positioned to function as mediators of communication between cancer cells and neurons. Inhibition of cancer-sEV release attenuated pain in tumor-bearing mice. Injection of purified cancer-sEVs is sufficient to induce pain hypersensitivity in naive mice that is prevented by QX-314 treatment and in Trpv1-/- mice. Cancer-sEVs triggered calcium influx in nociceptors, and inhibition or ablation of nociceptors protects against cancer pain. Interrogation of published sequencing data of human sensory neurons exposed to human cancer-sEVs suggested a stimulation of protein translation in neurons. Induction of translation by cancer-sEVs was validated in our mouse model, and its inhibition alleviated cancer pain in mice. In summary, our work reveals that HPV+ head and neck squamous cell carcinoma-derived sEVs alter TRPV1+ neurons by promoting nascent translation to mediate cancer pain and identified several promising therapeutic targets to interfere with this pathway.

Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants.

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Tonic Meningeal Interleukin-10 Upregulates Delta Opioid Receptor to Prevent Relapse to Pain.

Chronic pain often alternates between transient remission and relapse of severe pain. While most research on chronic pain has focused on mechanisms maintaining pain, there is a critical unmet need to understand what prevents pain from re-emerging in those who recover from acute pain. We found that interleukin (IL)-10, a pain resolving cytokine, is persistently produced by resident macrophages in the spinal meninges during remission from pain. IL-10 upregulated expression and analgesic activity of δ-opioid receptor (δOR) in the dorsal root ganglion. Genetic or pharmacological inhibition of IL-10 signaling or δOR triggered relapse to pain in both sexes. These data challenge the widespread assumption that remission of pain is simply a return to the naïve state before pain was induced. Instead, our findings strongly suggest a novel concept that: remission is a state of lasting pain vulnerability that results from a long-lasting neuroimmune interactions in the nociceptive system.

An engineered cell line with a hRpn1-attached handle to isolate proteasomes.

Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which contains a 19-subunit regulatory particle (RP) that binds, processes, and translocates substrates to a 28-subunit hollow core particle (CP) where proteolysis occurs. In addition to its intrinsic subunits, myriad proteins interact with the proteasome transiently, including factors that assist and/or regulate its degradative activities. Efforts to identify proteasome-interacting components and/or to solve its structure have relied on over-expression of a tagged plasmid, establishing stable cell lines, or laborious purification protocols to isolate native proteasomes from cells. Here, we describe an engineered human cell line, derived from colon cancer HCT116 cells, with a biotin handle on the RP subunit hRpn1/PSMD2 (proteasome 26S subunit, non-ATPase 2) for purification of 26S proteasomes. A 75-residue sequence from Propionibacterium shermanii that is biotinylated in mammalian cells was added following a tobacco etch virus protease cut site at the C terminus of hRpn1. We tested and found that 26S proteasomes can be isolated from this modified HCT116 cell line by using a simple purification protocol. More specifically, biotinylated proteasomes were purified from the cell lysates by using neutravidin agarose resin and released from the resin following incubation with tobacco etch virus protease. The purified proteasomes had equivalent activity in degrading a model ubiquitinated substrate, namely ubiquitinated p53, compared to commercially available bovine proteasomes that were purified by fractionation. In conclusion, advantages of this approach to obtain 26S proteasomes over others is the simple purification protocol and that all cellular proteins, including the tagged hRpn1 subunit, remain at endogenous stoichiometry.

Protonation-Dependent Sequencing of 5-Formylcytidine in RNA.

Chemical modification of cytidine in noncoding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remain unknown due to a lack of sensitive site-specific sequencing technologies. Here, we report a protonation-dependent sequencing reaction for the detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron-withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human transfer RNA (tRNA). Finally, we demonstrate the utility of our method to characterize a patient-derived model of 5fC deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3-dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA and more broadly provide a molecular foundation for using optimized sequencing reactions to better understand the role of oxidized RNA cytidine residues in diseases.

Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma.

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 ß-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. 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.

Prenatal auditory learning in avian vocal learners and non-learners.

Understanding when learning begins is critical for identifying the factors that shape both the developmental course and the function of information acquisition. Until recently, sufficient development of the neural substrates for any sort of vocal learning to begin in songbirds was thought to be reached well after hatching. New research shows that embryonic gene activation and the outcome of vocal learning can be modulated by sound exposure in ovo. We tested whether avian embryos across lineages differ in their auditory response strength and sound learning in ovo, which we studied in vocal learning (Maluridae, Geospizidae) and vocal non-learning (Phasianidae, Spheniscidae) taxa. While measuring heart rate in ovo, we exposed embryos to (i) conspecific or heterospecific vocalizations, to determine their response strength, and (ii) conspecific vocalizations repeatedly, to quantify cardiac habituation, a form of non-associative learning. Response strength towards conspecific vocalizations was greater in two species with vocal production learning compared to two species without. Response patterns consistent with non-associative auditory learning occurred in all species. Our results demonstrate a capacity to perceive and learn to recognize sounds in ovo, as evidenced by habituation, even in species that were previously assumed to have little, if any, vocal production learning. This article is part of the theme issue 'Vocal learning in animals and humans'.

Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response.

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. Although in vitro-activated MAIT cells show antitumor activity, the in vivo role of MAIT cells in cancer is still unclear. Here, we have shown that MAIT cells have antitumor function in vivo when activated by a combination of the synthetic riboflavin synthesis pathway-derived antigen 5-OP-RU [5-(2-oxopropylideneamino)-6-D-ribitylaminouracil] and the Toll-like receptor 9 (TLR9) agonist CpG. Coadministration of 5-OP-RU and CpG induced strong systemic in vivo expansion and activation of MAIT cells with high CD69 expression, pronounced effector memory phenotype, and upregulated levels of effector molecules including IFNγ, granzyme B, and perforin. Activated and expanded MAITs induced a potent and broad antitumor immune response in murine models of liver metastasis and hepatocellular carcinoma, lung metastasis, and subcutaneous tumors in two different mouse strains. Such tumor inhibition was absent in MAIT-deficient Mr1 -/- mice. CRISPR/Cas9-mediated MR1 knockout in tumor cells did not affect efficacy of this MAIT-directed immunotherapy, pointing toward an indirect mechanism of action. Our findings suggest that MAIT cells are an attractive target for cancer immunotherapy.See related Spotlight by Lantz, p. 996.

Small-Molecule Natural Product Physachenolide C Potentiates Immunotherapy Efficacy by Targeting BET Proteins.

Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17ß-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand-dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8-dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome. SIGNIFICANCE: These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3374/F1.large.jpg.

Sex role similarity and sexual selection predict male and female song elaboration and dimorphism in fairy-wrens.

Historically, bird song complexity was thought to evolve primarily through sexual selection on males; yet, in many species, both sexes sing and selection pressure on both sexes may be broader. Previous research suggests competition for mates and resources during short, synchronous breeding seasons leads to more elaborate male songs at high, temperate latitudes. Furthermore, we expect male-female song structure and elaboration to be more similar at lower, tropical latitudes, where longer breeding seasons and year-round territoriality yield similar social selection pressures in both sexes. However, studies seldom take both types of selective pressures and sexes into account. We examined song in both sexes in 15 populations of nine-fairy-wren species (Maluridae), a Southern Hemisphere clade with female song. We compared song elaboration (in both sexes) and sexual song dimorphism to latitude and life-history variables tied to sexual and social selection pressures and sex roles. Our results suggest that song elaboration evolved in part due to sexual competition in males: male songs were longer than female songs in populations with low male survival and less male provisioning. Also, female songs evolved independently of male songs: female songs were slower paced than male songs, although only in less synchronously breeding populations. We also found male and female songs were more similar when parental care was more equal and when male survival was high, which provides strong evidence that sex role similarity correlates with male-female song similarity. Contrary to Northern Hemisphere latitudinal patterns, male and female songs were more similar at higher, temperate latitudes. These results suggest that selection on song can be sex specific, with male song elaboration favored in contexts with stronger sexual selection. At the same time, selection pressures associated with sex role similarity appear to favor sex role similarity in song structure.

dbGuide: a database of functionally validated guide RNAs for genome editing in human and mouse cells.

With the technology's accessibility and ease of use, CRISPR has been employed widely in many different organisms and experimental settings. As a result, thousands of publications have used CRISPR to make specific genetic perturbations, establishing in itself a resource of validated guide RNA sequences. While numerous computational tools to assist in the design and identification of candidate guide RNAs exist, these are still just at best predictions and generally, researchers inevitably will test multiple sequences for functional activity. Here, we present dbGuide (https://sgrnascorer.cancer.gov/dbguide), a database of functionally validated guide RNA sequences for CRISPR/Cas9-based knockout in human and mouse. Our database not only contains computationally determined candidate guide RNA sequences, but of even greater value, over 4000 sequences which have been functionally validated either through direct amplicon sequencing or manual curation of literature from over 1000 publications. Finally, our established framework will allow for continual addition of newly published and experimentally validated guide RNA sequences for CRISPR/Cas9-based knockout as well as incorporation of sequences from different gene editing systems, additional species and other types of site-specific functionalities such as base editing, gene activation, repression and epigenetic modification.

Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity.

hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding but defective for proteasome interaction. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole-cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.

Coronavirus disease (COVID-19) Community Testing Team in Scotland: A 14-day review, 6 to 20 February 2020.

In response to the outbreak of COVID-19, we set up a team to carry out sampling in the community. This enabled individuals to remain in self-isolation in their own homes and to prevent healthcare settings and services from being overwhelmed by admissions for sampling of suspected cases. There is evidence that this is a cost effective, safe and necessary service to complement COVID-19 testing in hospitals.

Sensitivity of Mesothelioma Cells to PARP Inhibitors Is Not Dependent on BAP1 but Is Enhanced by Temozolomide in Cells With High-Schlafen 11 and Low-O6-methylguanine-DNA Methyltransferase Expression.

INTRODUCTION: BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. METHODS: A total of 10 patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone or in combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. RESULTS: BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 µM to greater than 50 µM and 0.039 µM to greater than 5 µM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. CONCLUSIONS: BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.

Evolution and effects of COVID-19 outbreaks in care homes: a population analysis in 189 care homes in one geographical region of the UK.

BACKGROUND: COVID-19 has affected care home residents internationally, but detailed information on outbreaks is scarce. We aimed to describe the evolution of outbreaks of COVID-19 in all care homes in one large health region in Scotland. METHODS: We did a population analysis of testing, cases, and deaths in care homes in the National Health Service (NHS) Lothian health region of the UK. We obtained data for COVID-19 testing (PCR testing of nasopharyngeal swabs for severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) and deaths (COVID-19-related and non-COVID-19-related), and we analysed data by several variables including type of care home, number of beds, and locality. Outcome measures were timing of outbreaks, number of confirmed cases of COVID-19 in care home residents, care home characteristics associated with the presence of an outbreak, and deaths of residents in both care homes and hospitals. We calculated excess deaths (both COVID-19-related and non-COVID-19-related), which we defined as the sum of deaths over and above the historical average in the same period over the past 5 years. FINDINGS: Between March 10 and Aug 2, 2020, residents at 189 care homes (5843 beds) were tested for COVID-19 when symptomatic. A COVID-19 outbreak was confirmed at 69 (37%) care homes, of which 66 (96%) were care homes for older people. The size of care homes for older people was strongly associated with a COVID-19 outbreak (odds ratio per 20-bed increase 3·35, 95% CI 1·99-5·63). 907 confirmed cases of SARS-CoV-2 infection were recorded during the study period, and 432 COVID-19-related deaths. 229 (25%) COVID-19-related cases and 99 (24%) COVID-related deaths occurred in five (3%) of 189 care homes, and 441 (49%) cases and 207 (50%) deaths were in 13 (7%) care homes. 411 (95%) COVID-19-related deaths occurred in the 69 care homes with a confirmed COVID-19 outbreak, 19 (4%) deaths were in hospital, and two (<1%) were in one of the 120 care homes without a confirmed COVID-19 outbreak. At the 69 care homes with a confirmed COVID-19 outbreak, 74 excess non-COVID-19-related deaths were reported, whereas ten non-COVID-19-related excess deaths were observed in the 120 care homes without a confirmed COVID-19 outbreak. 32 fewer non-COVID-19-related deaths than expected were reported among care home residents in hospital. INTERPRETATION: The effect of COVID-19 on care homes has been substantial but concentrated in care homes with known outbreaks. A key implication from our findings is that, if community incidence of COVID-19 increases again, many care home residents will be susceptible. Shielding care home residents from potential sources of SARS-CoV-2 infection, and ensuring rapid action to minimise outbreak size if infection is introduced, will be important for any second wave. FUNDING: None.

Genetic admixture predicts parasite intensity: evidence for increased hybrid performance in Darwin's tree finches.

Hybridization can increase adaptive potential when enhanced genetic diversity or novel genetic combinations confer a fitness advantage, such as in the evolution of anti-parasitic mechanisms. Island systems are especially susceptible to invasive parasites due to the lack of defence mechanisms that usually coevolve in long-standing host-parasite relationships. We test if host genetic admixture affects parasite numbers in a novel host-parasite association on the Galápagos Islands. Specifically, we compare the number of Philornis downsi in nests with offspring sired by Darwin's small tree finch (Camarhynchus parvulus), Darwin's medium tree finch (C. pauper) and hybrids of these two species. The number of P. downsi decreased with an increasing genetic admixture of the attending male, and nests of hybrid males had approximately 50% fewer parasites than C. parvulus nests, and approximately 60% fewer parasites than C. pauper nests. This finding indicates that hybridization in this system could be favoured by selection and reveal a mechanism to combat an invasive parasite.

Female in-nest chatter song increases predation.

Female song is an ancestral trait in songbirds, yet extant females generally sing less than males. Here, we examine sex differences in the predation cost of singing behaviour. The superb fairy-wren (Malurus cyaneus) is a Southern Hemisphere songbird; males and females provision the brood and produce solo song year-round. Both sexes had higher song rate during the fertile period and lower song rate during incubation and chick feeding. Females were more likely than males to sing close to or inside the nest. For this reason, female but not male song rate predicted egg and nestling predation. This study identifies a high fitness cost of song when a parent bird attends offspring inside a nest and explains gender differences in singing when there are gender differences in parental care.