Toxicity of cockroach gel baits to the oothecal parasitoid Aprostocetus hagenowii (Hymenoptera: Eulophidae) and implications for cockroach integrated pest management.

Aprostocetus hagenowii (Ratzeburg) is a parasitoid wasp that parasitizes the oothecae of peridomestic pest cockroaches. A. hagenowii has been used in integrated pest management (IPM) programs for cockroach control but little is known about how this parasitoid responds to the insecticides commonly used for cockroach management. Five insecticidal gel bait products containing indoxacarb, clothianidin, fipronil, dinotefuran, or abamectin B1 were tested for their toxicity towards A. hagenowii and the American cockroach, Periplaneta americana (L.; Blattodea: Blattidae), a host of A. hagenowii and a common pest. All baits were tested as fresh and 1-d aged deposits. Indoxacarb was the only active ingredient that did not cause significant (P < 0.05) A. hagenowii mortality compared to the control in both the fresh and aged gel experiments (Median survival time [MST]s: 168 h fresh, 72 h aged). Clothianidin caused the lowest A. hagenowii MSTs across experiments (24 h, fresh and aged). All baits caused significant P. americana mortality as fresh and 1-d aged deposits (P < 0.05). Indoxacarb appears most compatible with A. hagenowii in cockroach IPM.

Discovery of a 53BP1 Small Molecule Antagonist Using a Focused DNA-Encoded Library Screen.

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 µM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 µM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

Polymerase θ inhibition steps on the cGAS pedal.

Deficiencies in homologous recombination (HR) repair lead to an accumulation of DNA damage and can predispose individuals to cancer. Polymerase theta (Pol θ, encoded by POLQ) is overexpressed by HR-deficient cancers and promotes cancer cell survival by mediating error-prone double-stranded break (DSB) repair and facilitating resistance against poly-ADP ribose polymerase inhibitor treatment. In this issue of the JCI, Oh, Wang, et al. report on the impact of Pol θ inhibition on activation of antitumor immunity. The authors used pancreatic ductal adenocarcinoma (PDAC) cell and mouse models characterized by HR-associated gene alterations and POLQ overexpression. POLQ knockdown showed synthetic lethality in combination with gene mutations involving DNA repair, including BRCA1, BRCA2, and ATM. Notably, Pol θ deficiency or inhibition suppressed tumor growth, increased the accumulation of unrepaired DNA damage, and enhanced T cell infiltration via the cGAS/STING pathway. These findings suggest a broader scope for Pol θ inhibition in HR-deficient cancers.

Potential of the oothecal parasitoid Aprostocetus hagenowii (Hymenoptera: Eulophidae) as a biological control agent for the Turkestan cockroach (Blattodea: Blattidae).

The Turkestan cockroach, Blatta lateralis (Walker), is a peridomestic pest of growing concern in the US Southwest. The parasitoid Aprostocetus hagenowii (Ratzburg) is used in IPM programs targeting other blattid cockroach species and may aid in B. lateralis suppression. Information about the ability of A. hagenowii to parasitize B. lateralis is lacking. A no-choice host-switching experiment was used to test A. hagenowii acceptance of B. lateralis oothecae, and a multigenerational no-choice experiment was used to determine the suitability of B. lateralis as a host for A. hagenowii over several months of rearing. Periplaneta americana (L.) (Blattodea: Blattidae), the preferred host of A. hagenowii, and Blatta orientalis L., a known host and relative of B. lateralis, were used for comparison. Development time was similar among hosts and generations (P > 0.05). Parasitism success and proportion of female progeny declined significantly with subsequent generations on both Blatta spp. (parasitism success: χ2 = 14.916; df = 2; P = 0.001; proportion female: H = 6.364; df = 2; P = 0.041). These results suggest that A. hagenowii may initially aid in suppression of B. lateralis, but an overall decline in fitness will require repeated releases or provisioning of P. americana oothecae. Development of a strain more suitable for B. lateralis control may be possible via selection from laboratory strains or through use of wild A. hagenowii from areas where B. lateralis is present.

Kudzu Bug (Megacopta cribraria) and Associated Egg Parasitoids Emergence Rates in Alabama are Predicted by Weather Indices.

The kudzu bug, Megacopta cribraria (Fabricius) (Hemiptera: Plataspidae), is a major economic pest of soybean in the southeastern United States. With climate warming, this pest is expected to move northward and cause additional crop damage. Parasitoid biocontrol is a potential method of integrated pest management for kudzu bug. Two species of egg parasitoid wasps have been observed emerging from kudzu bug egg masses in the southeastern United States: Paratelenomus saccharalis (Dodd) (Hymenoptera: Platygastridae) and Ooencyrtus nezarae (Ishii) (Hymenoptera: Encyrtidae). This paper used egg mass emergence data collected between 2018 and 2020 in Alabama soybean fields and compared the data to weather indices. Indices included the number of days with minimum temperatures below zero, accumulated rainfall (mm m-2), as well as species specific metrics of accumulated growing degree days, accumulated daily minimum temperature (°C), and accumulated daily maximum temperature (°C). Emergence of the generalist parasitoid, O. nezarae, was highly correlated with kudzu bug nymph abundance, accumulated degree day, accumulated daily temperatures, and precipitation. Ooencyrtus nezarae emergence was predicted in a stepwise regression equation by aggregated degree day and date of collection, which indicates that seasonality may be a predictor of its presence. In contrast, collections of the specialist parasitoid, P. saccharalis, were near-zero throughout the collection period, suggesting that this species may no longer be a usable biocontrol agent in the southeastern United States as a result of external limiting factors.

Targeting Non-homologous and Alternative End Joining Repair to Enhance Cancer Radiosensitivity.

Many cancer therapies, including radiotherapy, induce DSBs as the major driving mechanism for inducing cancer cell death. Thus, modulating DSB repair has immense potential for radiosensitization, although such interventions must be carefully designed to be tumor selective to ensure that normal tissue toxicities are not also increased. Here, we review mechanisms of error-prone DSB repair through a highly efficient process called end joining. There are two major pathways of end-joining repair: non-homologous end joining (NHEJ) and alternative end joining (a-EJ), both of which can be selectively upregulated in cancer and thus represent attractive therapeutic targets for radiosensitization. These EJ pathways each have therapeutically targetable pioneer factors - DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ and DNA Polymerase Theta (Pol θ) for a-EJ. We summarize the current status of therapeutic targeting of NHEJ and a-EJ to enhance the effects of radiotherapy - focusing on challenges that must be overcome and opportunities that require further exploration. By leveraging preclinical insights into mechanisms of altered DSB repair programs in cancer, selective radiosensitization through NHEJ and/or a-EJ targeting remains a highly attractive avenue for ongoing and future clinical investigation.

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

The human Shu complex functions with PDS5B and SPIDR to promote homologous recombination.

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.

RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination.

The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.

Developmental and epigenetic effects of Roundup and glyphosate exposure on Japanese medaka (Oryzias latipes).

Roundup and other glyphosate-based herbicides are the most commonly used herbicides in the world, yet their effects on developing fish embryos are not clearly understood. The present study, therefore, examined developmental teratogenic effects and adult-onset reproductive effects of exposure to environmentally relevant concentrations of glyphosate and Roundup in Japanese medaka fish (Oryzias latipes). Hd-rR strain medaka embryos were exposed to 0.5 mg/L glyphosate, 0.5 mg/L and 5 mg/L Roundup (glyphosate acid equivalent) for the first 15 days of their embryonic life and then allowed to sexually mature without further exposure. Whole body tissue samples were collected at 15 days post fertilization (dpf) and brain and gonad samples were collected in mature adults. Hatching success and phenotypic abnormalities were recorded up until 15 dpf. Roundup (0.5 mg/L) and glyphosate decreased cumulative hatching success, while glyphosate exposure increased developmental abnormalities in medaka fry. Expression of the maintenance DNA methyltransferase gene Dnmt1 decreased, whereas expression of methylcytosine dioxygenase genes (Tet1, Tet2 and Tet3) increased in fry at 15 dpf suggesting that epigenetic alterations increased global DNA demethylation in the developing fry. Fecundity and fertilization efficiency were not altered due to exposure. Among the reproduction-related genes in the brain, kisspeptin receptor (Gpr54-1) expression was significantly reduced in females exposed to 0.5 mg/L and 5 mg/L Roundup, and Gpr54-2 was reduced in the 0.5 mg/L Roundup treatment group. No change in expression of these genes was observed in the male brain. In the testes, expression of Fshr and Arα was significantly reduced in medaka exposed to 0.5 mg/L Roundup and glyphosate, while the expression of Dmrt1 and Dnmt1 was reduced in medaka exposed to 0.5 mg/L glyphosate. No change in expression of these genes was observed in the ovaries. The present study demonstrates that Roundup and its active ingredient glyphosate can induce developmental, reproductive, and epigenetic effects in fish; suggesting that ecological species, mainly fish, could be at risk for endocrine disruption in glyphosate and Roundup-contaminated water bodies.