Therapeutic Delivery of Soluble Fractalkine Ameliorates Vascular Dysfunction in the Diabetic Retina.

Diabetic retinopathy (DR)-associated vision loss is a devastating disease affecting the working-age population. Retinal pathology is due to leakage of serum components into retinal tissues, activation of resident phagocytes (microglia), and vascular and neuronal damage. While short-term interventions are available, they do not revert visual function or halt disease progression. The impact of microglial inflammatory responses on the neurovascular unit remains unknown. In this study, we characterized microglia-vascular interactions in an experimental model of DR. Early diabetes presents activated retinal microglia, vascular permeability, and vascular abnormalities coupled with vascular tortuosity and diminished astrocyte and endothelial cell-associated tight-junction (TJ) and gap-junction (GJ) proteins. Microglia exclusively bind to the neuronal-derived chemokine fractalkine (FKN) via the CX3CR1 receptor to ameliorate microglial activation. Using neuron-specific recombinant adeno-associated viruses (rAAVs), we therapeutically overexpressed soluble (sFKN) or membrane-bound (mFKN) FKN using intra-vitreal delivery at the onset of diabetes. This study highlights the neuroprotective role of rAAV-sFKN, reducing microglial activation, vascular tortuosity, fibrin(ogen) deposition, and astrogliosis and supporting the maintenance of the GJ connexin-43 (Cx43) and TJ zonula occludens-1 (ZO-1) molecules. The results also show that microglia-vascular interactions influence the vascular width upon administration of rAAV-sFKN and rAAV-mFKN. Administration of rAAV-sFKN improved visual function without affecting peripheral immune responses. These findings suggest that overexpression of rAAV-sFKN can mitigate vascular abnormalities by promoting glia-neural signaling. sFKN gene therapy is a promising translational approach to reverse vision loss driven by vascular dysfunction.

Water availability and seasonality shape elemental stoichiometry across space and time.

The interaction of climate change and increasing anthropogenic water withdrawals is anticipated to alter surface water availability and the transport of carbon (C), nitrogen (N), and phosphorus (P) in river networks. But how changes to river flow will alter the balance, or stoichiometry, of these fluxes is unknown. The Lower Flint River Basin (LFRB) is part of an interstate watershed relied upon by several million people for diverse ecosystem services, including seasonal crop irrigation, municipal drinking water access, and public recreation. Recently, increased water demand compounded with intensified droughts have caused historically perennial streams in the LFRB to cease flowing, increasing ecosystem vulnerability. Our objectives were to quantify how riverine dissolved C:N:P varies spatially and seasonally and determine how monthly stoichiometric fluxes varied with overall water availability in a major tributary of LFRB. We used a long-term record (21-29 years) of solute water chemistry (dissolved organic carbon, nitrate/nitrite, ammonia, and soluble reactive phosphorus) paired with long-term stream discharge data across six sites within a single LFRB watershed. We found spatial and seasonal differences in soluble nutrient concentrations and stoichiometry attributable to groundwater connections, the presence of a major floodplain wetland, and flow conditions. Further, we showed that water availability, as indicated by the Palmer Drought Severity Index (PDSI), strongly predicted stoichiometry with generally lower C:N and C:P and higher N:P fluxes during periods of low water availability (PDSI < -4). These patterns suggest there may be long-term and significant changes to stream ecosystem function as water availability is being dramatically altered by human demand with consequential impacts on solute transport, in-stream processing, and stoichiometric ratios.

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.

Fitness of evolving bacterial populations is contingent on deep and shallow history but only shallow history creates predictable patterns.

Long-term evolution experiments have tested the importance of genetic and environmental factors in influencing evolutionary outcomes. Differences in phylogenetic history, recent adaptation to distinct environments and chance events, all influence the fitness of a population. However, the interplay of these factors on a population's evolutionary potential remains relatively unexplored. We tracked the outcome of 2000 generations of evolution of four natural isolates of Escherichia coli bacteria that were engineered to also create differences in shallow history by adding previously identified mutations selected in a separate long-term experiment. Replicate populations started from each progenitor evolved in four environments. We found that deep and shallow phylogenetic histories both contributed significantly to differences in evolved fitness, though by different amounts in different selection environments. With one exception, chance effects were not significant. Whereas the effect of deep history did not follow any detectable pattern, effects of shallow history followed a pattern of diminishing returns whereby fitter ancestors had smaller fitness increases. These results are consistent with adaptive evolution being contingent on the interaction of several evolutionary forces but demonstrate that the nature of these interactions is not fixed and may not be predictable even when the role of chance is small.

Seasonal Fluctuations in Iron Cycling in Thawing Permafrost Peatlands.

In permafrost peatlands, up to 20% of total organic carbon (OC) is bound to reactive iron (Fe) minerals in the active layer overlying intact permafrost, potentially protecting OC from microbial degradation and transformation into greenhouse gases (GHG) such as CO2 and CH4. During the summer, shifts in runoff and soil moisture influence redox conditions and therefore the balance of Fe oxidation and reduction. Whether reactive iron minerals could act as a stable sink for carbon or whether they are continuously dissolved and reprecipitated during redox shifts remains unknown. We deployed bags of synthetic ferrihydrite (FH)-coated sand in the active layer along a permafrost thaw gradient in Stordalen mire (Abisko, Sweden) over the summer (June to September) to capture changes in redox conditions and quantify the formation and dissolution of reactive Fe(III) (oxyhydr)oxides. We found that the bags accumulated Fe(III) under constant oxic conditions in areas overlying intact permafrost over the full summer season. In contrast, in fully thawed areas, conditions were continuously anoxic, and by late summer, 50.4 ± 12.8% of the original Fe(III) (oxyhydr)oxides were lost via dissolution. Periodic redox shifts (from 0 to +300 mV) were observed over the summer season in the partially thawed areas. This resulted in the dissolution and loss of 47.2 ± 20.3% of initial Fe(III) (oxyhydr)oxides when conditions are wetter and more reduced, and new formation of Fe(III) minerals (33.7 ± 8.6% gain in comparison to initial Fe) in the late summer under more dry and oxic conditions, which also led to the sequestration of Fe-bound organic carbon. Our data suggest that there is seasonal turnover of iron minerals in partially thawed permafrost peatlands, but that a fraction of the Fe pool remains stable even under continuously anoxic conditions.

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.

An Emergent Network for the Diffusion of Innovations Among Local Health Departments at the Onset of the COVID-19 Pandemic.

INTRODUCTION: Communication networks among professionals can be pathways for accelerating the diffusion of innovations if some local health departments (LHDs) drive the spread of knowledge. Such a network could prove valuable during public health emergencies such as the novel coronavirus disease 2019 (COVID-19) pandemic. Our objective was to determine whether LHDs in the United States were tied together in an informal network to share information and advice about innovative community health practices, programs, and policies. METHODS: In January and February 2020, we conducted an online survey of 2,303 senior LHD leaders to ask several questions about their sources of advice. We asked respondents to rank up to 3 other LHDs whose practices informed their work on new public health programs, evidence-based practices, and policies intended to improve community health. We used a social network analysis program to assess answers. RESULTS: A total of 329 LHDs responded. An emergent network appeared to operate nationally among 740 LHDs. Eleven LHDs were repeatedly nominated by peers as sources of advice or examples (ie, opinion leaders), and 24 acted as relational bridges to hold these emergent networks together (ie, boundary spanners). Although 2 LHDs played both roles, most LHDs we surveyed performed neither of these roles. CONCLUSION: Opinion leading and boundary spanning health departments can be accessed to increase the likelihood of affecting the rate of interest in and adoption of innovations. Decision makers involved in disseminating new public health practices, programs, or policies may find our results useful both for emergencies and for practice-as-usual.

Neurological Involvement in Glycogen Storage Disease Type IXa due to PHKA2 Mutation.

Glycogen storage diseases (GSDs) result from the deficiency of enzymes involved in glycogen synthesis and breakdown into glucose. Mutations in the gene PHKA2 encoding phosphorylase kinase regulatory subunit alpha 2 have been linked to GSD type IXa. We describe a family with two adult brothers with neonatal hepatosplenomegaly and later onset of hearing loss, cognitive impairment, and cerebellar involvement. Whole-exome sequencing was performed on both subjects and revealed a shared hemizygous missense variant (c.A1561G; p.T521A) in exon 15 of PHKA2. The phenotype broadens the clinical and magnetic resonance imaging spectrum of GSD type IXa to include later onset neurological manifestations.

Family Instability and Exposure to Violence in the Early Life Course.

Family instability has been linked with a host of outcomes across the early life course. This study extends this literature by connecting instability with violence in the community by examining the associations among family structure, family structure change, and secondary exposure to violence during adolescence across diverse segments of the population. Using longitudinal data from the Project on Human Development in Chicago Neighborhoods study, we found that living with a single parent and experiencing family structure changes were associated with secondary exposure to violence. Multiple group models suggest that partner change translated into more exposure for boys than girls. Findings also suggest that family instability may lead to more secondary exposure to violence for African American youth.

Family, Academic, and Peer Group Predictors of Adolescent Pregnancy Expectations and Young Adult Childbearing.

Compared with previous generations, today's young people increasingly delay parenthood. Having children in the late teens and early 20s is thus a rarer experience rooted in and potentially leading to the stratification of American families. Understanding why some adolescents expect to do so can illuminate how stratification unfolds. Informed by theories of the life course, social control, and reasoned action, this study used the National Longitudinal Survey of Youth 1997 cohort (n = 4,556) to explore outcomes and antecedents of adolescent pregnancy expectations with logistic regressions. Results indicated that those expectations-including neither low nor high (i.e., split) expectations-predicted subsequent childbearing. These apparently consequential expectations were, in turn, most closely associated with youth's academics and peer groups. These findings illustrate how different domains can intersect in the early life course to shape future prospects, and they emphasize split pregnancy expectations reported in a nationally representative sample of young women and men.

Family-related Disparities in College Enrollment across the Great Recession.

The economic crisis of the Great Recession in the late 2000s had implications for the intergenerational transmission of inequality within families. Studying patterns of college enrollment across the Great Recession among U.S. youth from diverse family contexts provides insight into how economic volatility can either compound or undercut the advantages that some parents can give their children. Although college enrollment among 18- to 21-year-olds did not decline during or after the Great Recession, analyses of the National Longitudinal Survey of Youth 1979-Young Adult cohort revealed that this general trend subsumed variability by family history, local economic conditions, and age. Histories of family stability and sufficiency were associated with higher odds of college enrollment over time and across age, but this advantage was largest during the Recession in high-unemployment communities. These results illuminate how life course consequences of early family life can fluctuate with volatility and opportunity in the broader economy.

Differential control of opioid antinociception to thermal stimuli in a knock-in mouse expressing regulator of G-protein signaling-insensitive Gαo protein.

Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the µ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, Gαo(G184S) (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.

Changes in inundation drive carbon dioxide and methane fluxes in a temperate wetland.

Wetlands cycle carbon by being net sinks for carbon dioxide (CO2) and net sources of methane (CH4). Daily and seasonal temporal patterns, dissolved oxygen (DO) availability, inundation status (flooded or dry/partially flooded), water depth, and vegetation can affect the magnitude of carbon uptake or emissions, but the extent and interactive effects of these variables on carbon gas fluxes are poorly understood. We characterized the linkages between carbon fluxes and these environmental and temporal drivers at the Old Woman Creek National Estuarine Research Reserve (OWC), OH. We measured diurnal gas flux patterns in an upstream side channel (called the cove) using chamber measurements at six sites (three vegetated and three non-vegetated). We sampled hourly from 7 AM to 7 PM and monthly from July to October 2022. DO concentrations and water levels were measured monthly. Water inundation status had the most influential effect on carbon fluxes with flooded conditions supporting higher CH4 fluxes (0.39 µmol CH4 m-2 s-1; -1.23 µmol CO2 m-2 s-1) and drier conditions supporting higher CO2 fluxes (0.03 µmol CH4 m-2 s-1; 0.86 µmol CO2 m-2 s-1). When flooded, the wetland was a net CO2 sink; however, it became a source for both CH4 and CO2 when water levels were low. We compared chamber-based gas fluxes from the cove in flooded (July) and dry (August) months to fluxes measured with an eddy covariance tower whose footprint covers flooded portions of the wetland. The diurnal pattern of carbon fluxes at the tower did not vary with changing water levels but remained a CO2 sink and a CH4 source even when the cove where we performed the chamber measurements dried out. These results emphasize the role of inundation status on wetland carbon cycling and highlight the importance of fluctuating hydrologic patterns, especially hydrologic drawdowns, under changing climatic conditions.

The human Shu complex promotes RAD51 activity by modulating RPA dynamics on ssDNA.

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1 and SWS1. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter SWS1 complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.

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.

Impact of a 5-Week Individualised Training Program on Physical Performance and Measures Associated with Musculoskeletal Injury Risk in Army Personnel: A Pilot Study.

OBJECTIVE: To examine the feasibility and effect of an individualised and force-plate guided training program on physical performance and musculoskeletal injury risk factors in army personnel. DESIGN: Pre-post, randomised control. METHODS: Fourteen male and five female Australian Army soldiers were randomised into two groups and performed 5-weeks of physical training. The control group (n = 9) completed standard, group-designed, physical training whilst the experimental group (n = 8) completed an individualised training program. Physical (push-ups, multi-stage fitness test, three repetition maximum (3RM) for squat, strict press, deadlift and floor press), occupational (weight-loaded march time), and technological assessments (two-leg and one-leg countermovement jumps (CMJ), one-leg balance, one-arm plank) were conducted prior to and following the training period. Comparisons between groups and changes within groups were conducted via Mann-Whitney U tests. RESULTS: Compared to the control group, the experimental group exhibited a significantly smaller improvement for weight-loaded march time (-0.7% ± 4.0% vs. -5.1% ± 3.0%, p = 0.03) and a greater improvement for deadlift-3RM (20.6% ± 11.9% vs. 8.4% ± 6.8%, p = 0.056). All other outcomes were similar between groups. Visually favourable alterations in the two-leg CMJ profile with no reports of injuries were noted for the experimental group. CONCLUSIONS: Individualised physical training was feasible within an army setting and, for the most part, produced similar physical, occupational and technological performances to that of standard, group-designed physical training. These preliminary results provide a foundation for future research to expand upon and clarify the benefits of individualised training programs on long-term physical performance and injury risk/incidence in active combat army personnel.

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.

Exploring experiences of loneliness among Canadian long-term care residents during the COVID-19 pandemic: A qualitative study.

BACKGROUND: The COVID-19 pandemic has significant impact on long-term care (LTC) residents' health and well-being. OBJECTIVES: This study investigated resident experiences of loneliness during the COVID-19 pandemic in Canadian LTC homes to offer lessons learned and implications. METHODS: 15 residents and 16 staff members were recruited from two large urban Canadian LTC homes with large outbreaks and fatalities. We used a telepresence robot to conduct one-on-one semi-structured interviews with participants remotely. We applied the Collaborative Action Research (CAR) methodology and report the early phase of CAR focused on collecting data and reporting findings to inform actions for change. Thematic analysis was performed to identify themes. RESULTS: Four themes were identified. The first two themes characterise what commonly generated feelings of loneliness amongst residents, including (1) social isolation and missing their family and friends and (2) feeling hopeless and grieving for lives lost. The second two themes describe what helped residents alleviate loneliness, including (3) social support and (4) creating opportunities for recreation and promoting positivity. CONCLUSIONS: Residents living in LTC experienced significant social isolation and grief during the pandemic that resulted in loneliness and other negative health consequences. IMPLICATIONS FOR PRACTICE: Promoting meaningful connection, safe recreational activities and a positive atmosphere in LTC homes during the pandemic may help mitigate residents' experiences of loneliness due to social isolation and/or grief and enhance their quality of life.

Team science: A syllabus for success on big projects.

Interdisciplinary teams are on the rise as scientists attempt to address complex environmental issues. While the benefits of team science approaches are clear, researchers often struggle with its implementation, particularly for new team members. The challenges of large projects often weigh on the most vulnerable members of a team: trainees, including undergraduate students, graduate students, and post-doctoral researchers. Trainees on big projects have to navigate their role on the team, with learning project policies, procedures, and goals, all while also training in key scientific tasks such as co-authoring papers. To address these challenges, we created and participated in a project-specific, graduate-level team science course. The purposes of this course were to: (1) introduce students to the goals of the project, (2) build trainees' understanding of how big projects operate, and (3) allow trainees to explore how their research interests dovetailed with the overall project. Additionally, trainees received training regarding: (1) diversity, equity & inclusion, (2) giving and receiving feedback, and (3) effective communication. Onboarding through the team science course cultivated psychological safety and a collaborative student community across disciplines and institutions. Thus, we recommend a team science course for onboarding students to big projects to help students establish the skills necessary for collaborative research. Project-based team science classes can benefit student advancement, enhance the productivity of the project, and accelerate the discovery of solutions to ecological issues by building community, establishing a shared project vocabulary, and building a workforce with collaborative skills to better answer ecological research questions.

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.