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Abstract Gene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient‐specific skin equivalents. Precise gene editing using homology‐directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress. Reframing strategies based on highly efficient non‐homologous end joining (NHEJ) repair aimed at excising dispensable, mutation‐harboring exons offer a promising alternative approach for restoring the COL7A1 open reading frame. To this end, we employed an exon skipping strategy using dual single guide RNA (sgRNA)/Cas9 ribonucleoproteins (RNPs) targeted at three novel COL7A1 exons (31, 68, and 109) containing pathogenic heterozygous mutations, and achieved exon deletion rates of up to 95%. Deletion of exon 31 in both primary human RDEB keratinocytes and fibroblasts resulted in the restoration of type VII collagen (C7), leading to increased cellular adhesion in vitro and accurate C7 deposition at the dermal‐epidermal junction in a 3D skin model. Taken together, we extend the list of COL7A1 exons amenable to therapeutic deletion. As an incidental finding, we find that long‐read Nanopore sequencing detected large on‐target structural variants comprised of deletions up to >5 kb at a frequency of ~10%. Although this frequency may be acceptable given the high rates of intended editing outcomes, our data demonstrate that standard short‐read sequencing may underestimate the full range of unexpected Cas9‐mediated editing events.

Abstract Background Considering animals as individuals and not as species is becoming increasingly essential to animal welfare management in captive settings. Recent studies on big cat personalities and coping strategies suggest personality can help big cats cope in their surroundings. Yet a large portion of the published literature focuses on understanding either the personality or stress physiology of big cats. Our research shows how integrating an improved understanding of the personality of big cats with stress physiology may enhance welfare, especially for endangered species like African lions. By using a wild cat personality checklist, this study compared the key personality dimensions of 22 African lions with its faecal glucocorticoids and assessed factors influencing their personality and stress physiology. Results We found two reliable personality dimensions for African lions (dominance and agreeableness) and identified key factors (sex, age and location) that may influence their personality. Further, on testing if these factors influenced the stress physiology through variations in glucocorticoid levels, there was no significant difference. However, there was a strong negative association between agreeableness and glucocorticoid levels. These results suggest that the behavioural traits loading positively and higher for agreeableness are associated with lower glucocorticoid stress levels, which may assist a lion to cope with stressors in its surroundings. Conclusions Our findings highlight this integrated approach of linking personality and stress physiology of big cats can be beneficial for caretakers. For example, during stressful veterinary procedures or in reintroduction programs, recognizing the personality of lions can help in designing or providing them with resources that will alleviate stress. Thus, there is a need for more interdisciplinary approaches that will contribute towards enhancing the individual and overall welfare of big cats.

Background: Intensified training coupled with sufficient recovery is required to improve athletic performance. A stress-recovery imbalance can lead to negative states of overtraining. Hormonal alterations associated with intensified training, such as blunted cortisol, may impair the immune response. Cortisol promotes the maturation and migration of dendritic cells which subsequently stimulate the T cell response. However, there are currently no clear reliable biomarkers to highlight the overtraining syndrome. This systematic review and meta-analysis examined the effect of intensified training on immune cells. Outcomes from this could provide insight into whether these markers may be used as an indicator of negative states of overtraining.Methods: SPORTDiscus, PUBMED, Academic Search Complete, Scopus and Web of Science were searched until June 2022. Included articles reported on immune biomarkers relating to lymphocytes, dendritic cells, and cytokines before and after a period of intensified training, in humans and rodents, at rest and in response to exercise.Results: 164 full texts were screened for eligibility. Across 57 eligible studies, 16 immune biomarkers were assessed. 7 were assessed at rest and in response to a bout of exercise, and 9 assessed at rest only. Included lymphocyte markers were CD3+, CD4+ and CD8+ T cell count, NK cell count, NK Cytolytic activity, lymphocyte proliferation and CD4/CD8 ratio. Dendritic cell markers examined were CD80, CD86, and MHC II expression. Cytokines included IL-1β, IL-2, IL-10, TNF-α and IFN-γ. A period of intensified training significantly decreased resting total lymphocyte (d= −0.57, 95% CI −0.30) and CD8+ T cell counts (d= −0.37, 95% CI −0.04), and unstimulated plasma IL-1β levels (d= −0.63, 95% CI −0.17). Resting dendritic cell CD86 expression significantly increased (d = 2.18, 95% CI 4.07). All other biomarkers remained unchanged.Conclusion: Although some biomarkers alter after a period of intensified training, definitive immune biomarkers are limited. Specifically, due to low study numbers, further investigation into the dendritic cell response in human models is required.

Background Patient selection for resuscitative endovascular balloon occlusion of the aorta (REBOA) has evolved during the last decade. A recent multicenter collaboration to implement the newest generation REBOA balloon catheter identified variability in patient selection criteria. The aims of this systematic review were to compare recent REBOA patient selection guidelines and to identify current areas of consensus and variability.Methods In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, we conducted a systematic review of clinical practice guidelines for REBOA patient selection in trauma. Published algorithms from 2015 to 2022 and institutional guidelines from a seven-center REBOA collaboration were compiled and synthesized.Results Ten published algorithms and seven institutional guidelines on REBOA patient selection were included. Broad consensus exists on REBOA deployment for blunt and penetrating trauma patients with non-compressible torso hemorrhage refractory to blood product resuscitation. Algorithms diverge on precise systolic blood pressure triggers for early common femoral artery access and REBOA deployment, as well as the use of REBOA for traumatic arrest and chest or extremity hemorrhage control.Conclusion Although our convenience sample of institutional guidelines likely underestimates patient selection variability, broad consensus exists in the published literature regarding REBOA deployment for blunt and penetrating trauma patients with hypotension not responsive to resuscitation. Several areas of patient selection variability reflect individual practice environments.Level of evidence Level 5, systematic review.

Abstract Nutritional geometry has advanced our understanding of how macronutrients (e.g., proteins and carbohydrates) influence the expression of life history traits and their corresponding trade‐offs. For example, recent work has revealed that reproduction and immune function in male decorated crickets are optimized at very different protein:carbohydrate (P:C) dietary ratios. However, it is unclear how an individual's macronutrient intake interacts with its perceived infection status to determine investment in reproduction or other key life history traits. Here, we employed a fully factorial design in which calling effort and immune function were quantified for male crickets fed either diets previously demonstrated to maximize calling effort (P:C = 1:8) or immune function (P:C = 5:1), and then administered a treatment from a spectrum of increasing infection cue intensity using heat‐killed bacteria. Both diet and a simulated infection threat independently influenced the survival, immunity, and reproductive effort of males. If they called, males increased calling effort at the low infection cue dose, consistent with the terminal investment hypothesis, but interpretation of responses at the higher threat levels was hampered by the differential mortality of males across infection cue and diet treatments. A high protein, low carbohydrate diet severely reduced the health, survival, and overall fitness of male crickets. There was, however, no evidence of an interaction between diet and infection cue dose on calling effort, suggesting that the threshold for terminal investment was not contingent on diet as investigated here.

Abstract Evolutionary adaptation is generally thought to occur through incremental mutational steps, but large mutational leaps can occur during its early stages. These are challenging to study in nature due to the difficulty of observing new genetic variants as they arise and spread, but characterizing their genomic dynamics is important for understanding factors favoring rapid adaptation. Here, we report genomic consequences of recent, adaptive song loss in a Hawaiian population of field crickets (Teleogryllus oceanicus). A discrete genetic variant, flatwing, appeared and spread approximately 15 years ago. Flatwing erases sound‐producing veins on male wings. These silent flatwing males are protected from a lethal, eavesdropping parasitoid fly. We sequenced, assembled and annotated the cricket genome, produced a linkage map, and identified a flatwing quantitative trait locus covering a large region of the X chromosome. Gene expression profiling showed that flatwing is associated with extensive genome‐wide effects on embryonic gene expression. We found that flatwing male crickets express feminized chemical pheromones. This male feminizing effect, on a different sexual signaling modality, is genetically associated with the flatwing genotype. Our findings suggest that the early stages of evolutionary adaptation to extreme pressures can be accompanied by greater genomic and phenotypic disruption than previously appreciated, and highlight how abrupt adaptation might involve suites of traits that arise through pleiotropy or genomic hitchhiking.

Interest in developing food, feed, and other useful products from farmed insects has gained remarkable momentum in the past decade. Crickets are an especially popular group of farmed insects due to their nutritional quality, ease of rearing, and utility. However, production of crickets as an emerging commodity has been severely impacted by entomopathogenic infections, about which we know little. Here, we identified and characterized an unknown entomopathogen causing mass mortality in a lab-reared population of Gryllodes sigillatus crickets, a species used as an alternative to the popular Acheta domesticus due to its claimed tolerance to prevalent entomopathogenic viruses. Microdissection of sick and healthy crickets coupled with metagenomics-based identification and real-time qPCR viral quantification indicated high levels of cricket iridovirus (CrIV) in a symptomatic population, and evidence of covert CrIV infections in a healthy population. Our study also identified covert infections of Acheta domesticus densovirus (AdDNV) in both populations of G. sigillatus. These results add to the foundational research needed to better understand the pathology of mass-reared insects and ultimately develop the prevention, mitigation, and intervention strategies needed for economical production of insects as a commodity.

Despite decades of focus on crickets (family: Gryllidae) as a popular commodity and model organism, we still know very little about their immune responses to microbial pathogens. Previous studies have measured downstream immune effects (e.g., encapsulation response, circulating hemocytes) following an immune challenge in crickets, but almost none have identified and quantified the expression of immune genes during an active pathogenic infection. Furthermore, the prevalence of covert (i.e., asymptomatic) infections within insect populations is becoming increasingly apparent, yet we do not fully understand the mechanisms that maintain low viral loads. In the present study, we measured the expression of several genes across multiple immune pathways in Gryllodes sigillatus crickets with an overt or covert infection of cricket iridovirus (CrIV). Crickets with overt infections had higher relative expression of key pathway component genes across the Toll, Imd, Jak/STAT, and RNAi pathways. These results suggests that crickets can tolerate low viral infections but can mount a robust immune response during an overt CrIV infection. Moreover, this study provides insight into the immune strategy of crickets following viral infection and will aid future studies looking to quantify immune investment and improve resistance to pathogens.

The oxidative stress theory predicts that the accumulation of oxidative damage causes aging. More generally, oxidative damage could be a cost of reproduction that reduces survival. Both of these hypotheses have mixed empirical support. To better understand the life-history consequences of oxidative damage, we fed male and female Australian field crickets (Teleogryllus commodus) four diets differing in their protein and carbohydrate content, which have sex-specific effects on reproductive effort and lifespan. We supplemented half of these crickets with the vitamin E isoform DL-alpha-tocopherol and measured the effects of nutrient intake on lifespan, reproduction, oxidative damage and antioxidant protection. We found a clear trade-off between reproductive effort and lifespan in females but not in males. In direct contrast to the oxidative stress theory, crickets fed diets that improved their lifespan had high levels of oxidative damage to proteins. Supplementation with DL-alpha-tocopherol did not significantly improve lifespan or reproductive effort. However, males fed diets that increased their reproductive investment experienced high oxidative damage to proteins. While this suggests that male reproductive effort could elevate oxidative damage, this was not associated with reduced male survival. Overall, these results provide little evidence that oxidative damage plays a central role in mediating life-history trade-offs in T. commodus.

The Luneburg lens is a powerful imaging device, exhibiting aberration free focusing for parallel rays incident from any direction. However, its advantages are offset by a focal surface that is spherical and thus difficult to integrate with standard planar detector and emitter arrays. Using the recently developed technique of transformation optics, it is possible to transform the curved focal surface to a flat plane while maintaining the perfect focusing behavior of the Luneburg over a wide field of view. Here we apply these techniques to a lesser-known refractive Luneburg lens and implement the design with a metamaterial composed of a semi-crystalline distribution of holes drilled in a dielectric. In addition, we investigate the aberrations introduced by various approximations made in the implementation of the lens. The resulting design approach has improved mechanical strength with small aberrations and is ideally suited to implementation at infrared and visible wavelengths.

Nuptial gifts produced by males and transferred to females during copulation are common in insects. Yet, their precise composition and subsequent physiological effects on the female recipient remain unresolved. Male decorated crickets Gryllodes sigillatus transfer a spermatophore to the female during copulation that is composed of an edible gift, the spermatophylax, and the ampulla that contains the ejaculate. After transfer of the spermatophore, the female detaches the spermatophylax and starts to eat it while sperm from the ampulla are evacuated into the female reproductive tract. When the female has finished consuming the spermatophylax, she detaches the ampulla and terminates sperm transfer. Hence, one simple function of the spermatophylax is to ensure complete sperm transfer by distracting the female from prematurely removing the ampulla. However, the majority of orally active components of the spermatophylax itself and their subsequent effects on female behavior have not been identified. Here, we report the first analysis of the proteome of the G. sigillatus spermatophylax and the transcriptome of the male accessory glands that make these proteins. The accessory gland transcriptome was assembled into 17,691 transcripts whilst about 30 proteins were detected within the mature spermatophylax itself. Of these 30 proteins, 18 were encoded by accessory gland encoded messages. Most spermatophylax proteins show no similarity to proteins with known biological functions and are therefore largely novel. A spermatophylax protein shows similarity to protease inhibitors suggesting that it may protect the biologically active components from digestion within the gut of the female recipient. Another protein shares similarity with previously characterized insect polypeptide growth factors suggesting that it may play a role in altering female reproductive physiology concurrent with fertilization. Characterization of the spermatophylax proteome provides the first step in identifying the genes encoding these proteins in males and in understanding their biological functions in the female recipient.

Rapid and divergent evolution of male genital morphology is a conspicuous and general pattern across internally fertilizing animals. Rapid genital evolution is thought to be the result of sexual selection, and the role of natural selection in genital evolution remains controversial. However, natural and sexual selection are believed to act antagonistically on male genital form. We conducted an experimental evolution study to investigate the combined effects of natural and sexual selection on the genital-arch lobes of male Drosophila simulans. Replicate populations were forced to evolve under lifetime monogamy (relaxed sexual selection) or lifetime polyandry (elevated sexual selection) and two temperature regimes, 25°C (relaxed natural selection) or 27°C (elevated natural selection) in a fully factorial design. We found that natural and sexual selection plus their interaction caused genital evolution. Natural selection caused some aspects of genital form to evolve away from their sexually selected shape, whereas natural and sexual selection operated in the same direction for other shape components. Additionally, sexual and natural selection tended to favour larger genitals. Thus we find that the underlying selection driving genital evolution is complex, does not only involve sexual selection, and that natural selection and sexual selection do not always act antagonistically.

Recent research has highlighted the potential importance of environmental and genotype-by-environment (G×E) variation in sexual selection, but most studies have focussed on the expression of male sexual traits. Consequently, our understanding of genetic variation for plasticity in female mate choice is extremely poor. In this study we examine the genetics of female mate choice in Drosophila simulans using isolines reared across two post-eclosion temperatures. There was evidence for G×Es in female choosiness and preference, which suggests that the evolution of female mate choice behaviour could differ across environments. However, the ranked order of preferred males was consistent across females and environments, so the same males are favoured by mate choice in spite of G×Es. Our study highlights the importance of taking cross-environment perspectives in order to gain a more comprehensive understanding of the operation of sexual selection.

The immunocompetence handicap hypothesis predicts that male sexual trait expression should be positively correlated with immunocompetence. Here we investigate if immune function in the cricket, Teleogryllus commodus, is related to specific individual components of male sexual signals, as well as to certain multivariate combinations of these components that females most strongly prefer. Male T. commodus produce both advertisement and courtship calls prior to mating. We measured fine-scale structural parameters of both call types and also recorded nightly advertisement calling effort. We then measured two standard indices of immune function: lysozyme-like activity of the haemolymph and haemocyte counts. We found a weak, positive relationship between advertisement calling effort and lysozyme-like activity. There was, however, little evidence that individual structural call components or the net multivariate attractiveness of either call type signalled immune function. The relationships between immunity and sexual signaling did not differ between inbred and outbred males. Our data suggest that it is unlikely that females assess overall male immune function using male calls.

Studies that assess the success of riparian restoration projects seldom focus on wildlife. More generally, vegetation characteristics are studied, with the assumption that animal populations will recover once adequate habitats are established. On the Sacramento River, millions of dollars have been spent on habitat restoration, yet few studies of wildlife response have been published. Here we present the major findings of a suite of studies that assessed responses of four taxonomic groups (insects, birds, bats, and rodents). Study designs fell primarily into two broad categories: comparisons of restoration sites of different ages, and comparisons of restoration sites with agricultural and remnant riparian sites. Older restoration sites showed increased abundances of many species of landbirds and bats relative to younger sites, and the same trend was observed for the Valley elderberry longhorn beetle (Desmocerus californicus dimorphus), a federally threatened species. Species richness of landbirds and ground-dwelling beetles appeared to increase as restoration sites matured. Young restoration sites provided benefits to species that utilize early successional riparian habitats, and after about 10 years, the sites appeared to provide many of the complex structural habitat elements that are characteristic of remnant forest patches. Eleven-year old sites were occupied by both cavity-nesting birds and special-status crevice-roosting bats. Restored sites also supported a wide diversity of bee species, and had richness similar to remnant sites. Remnant sites had species compositions of beetles and rodents more similar to older sites than to younger sites. Because study durations were short for all but landbirds, results should be viewed as preliminary. Nonetheless, in aggregate, they provide convincing evidence that restoration along the Sacramento River has been successful in restoring riparian habitats for a broad suite of faunal species. Not only did the restoration projects provide benefits for special-status species, but they also appeared effective in restoring the larger native riparian community. Increases in bird abundance through time were observed both at restoration sites and in remnant habitats, suggesting that restoration efforts may be having positive spill-over effects, although observed increases may have been caused by other factors. Although positive overall, these studies yielded some disconcerting results. The Lazuli Bunting (Passerina amoena) declined at restoration sites and remnant habitats alike, and certain exotic invasive species, such as black rats, appeared to increase as restoration sites matured.

Differences in the ways in which males and females maximize evolutionary fitness can lead to intra-locus sexual conflict in which genes delivering fitness benefits to one sex are costly when expressed in the other. Trade-offs between current reproductive effort and future reproduction and survival are fundamental to the evolutionary biology of ageing. This leads to the prediction that sex differences in the optimization of age-dependent reproductive effort may generate intra-locus sexual conflict over ageing rates. Here we test for intra-locus sexual conflict over age-dependent reproductive effort and longevity in the black field cricket, Teleogryllus commodus. Using a half-sib breeding design, we show that the most important components of male and female reproductive effort (male calling effort and the number of eggs laid by females) were positively genetically correlated, especially in early adulthood. However, the genetic relationships between longevity and reproductive effort were different for males and females, leading to low genetic covariation between male and female longevity. The apparent absence of intra-locus sexual conflict over ageing suggests that male and female longevity can evolve largely independently of one another.

The fitness consequences of mate choice are a source of ongoing debate in evolutionary biology. Recent theory predicts that indirect benefits of female choice due to offspring inheriting superior genes are likely to be negated when there are direct costs associated with choice, including any costs of mating with attractive males. To estimate the fitness consequences of mating with males of varying attractiveness, we housed female house crickets, Acheta domesticus, with either attractive or unattractive males and measured a variety of direct and indirect fitness components. These fitness components were combined to give relative estimates of the number of grandchildren produced and the intrinsic rate of increase (relative net fitness). We found that females mated to attractive males incur a substantial survival cost. However, these costs are cancelled out and may be outweighed by the benefits of having offspring with elevated fitness. This benefit is due predominantly, but not exclusively, to the effect of an increase in sons' attractiveness. Our results suggest that the direct costs that females experience when mating with attractive males can be outweighed by indirect benefits. They also reveal the value of estimating the net fitness consequences of a mating strategy by including measures of offspring quality in estimates of fitness.

In this article, the author talks about his Dakota ancestry. Here, he also talks about "Lamenting Dakota Language Loss," a paper he delivered at the 2004 Modern Language Association (MLA) convention. At the convention, he raised several questions regarding the revitalization of endangered Native American languages. Among other things, he points out what the association urged colleges and universities should do: (1) teach the languages of Native American nations in their regions; (2) grant credit for the study of Native American language when undertaken to fulfill undergraduate and graduate requirements in foreign languages; (3) include, where appropriate, Native American languages in the curriculum in the same manner as foreign languages; (4) grant proficiency in Native American languages the same full academic credit as proficiency in foreign languages; (5) encourage research to create and update dictionaries, grammars, orthographies, curricula, and other materials to support the teaching of Native American languages...especially...languages for which they have never been developed; and (6) work with Native American language communities and with Native American educational and governing bodies to implement these recommendations. (Contains 27 notes.)