The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency

A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard

[EN] Background: The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information. Results: Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA con¿ struction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. Conclusions: The functionality and the efficiency of gRNA¿Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA¿Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineeringThis work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools.Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernández Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. https://doi.org/10.1186/s13007-016-0101-2S12Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 .Yang X. Applications of CRISPR-Cas9 mediated genome engineering. Mil Med Res. 2015;2:11. doi: 10.1186/s40779-015-0038-1 .Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. 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PLoS ONE. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553 .Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS ONE. 2011;6(7):e21622. doi: 10.1371/journal.pone.0021622 .Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014;7(9):1494–6. doi: 10.1093/mp/ssu044 .Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. doi: 10.1126/science.1232033 .Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31(8):688–91. doi: 10.1038/nbt.2654 .Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. 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Androgen and glucocorticoid levels reflect seasonally occurring social challenges in male redfronted lemurs (Eulemur fulvus rufus)

Intense reproductive competition and social instability are assumed to increase concentrations of glucocorticoids and androgens in vertebrates, as a means of coping with these challenges. In seasonally breeding redfronted lemurs (Eulemur fulvus rufus), the mating and the birth season and the associated increased male competition are predicted to pose such reproductive challenges. In this paper, we investigate seasonal variation in hormone excretion in male redfronted lemurs, and examine whether this variation is associated with social or ecological factors. Although dominance status has been shown to affect individual stress levels across many taxa, we predicted no rank-related differences in glucocorticoids for redfronted lemurs because relatively equal costs are associated with both high and low rank positions (based on patterns of rank acquisition/maintenance and threats toward subordinates). Over a 14-month period, we collected behavioral data (1843 focal hours) and 617 fecal samples from 13 redfronted lemur males in Kirindy Forest/Madagascar. We found no general rank-related pattern of testosterone or glucocorticoid excretion in this species. Both hormones were excreted at significantly higher levels during the mating and the birth season, despite social stability during both periods. The elevated mating season levels may be explained by increased within-group reproductive competition during this time and are in line with previous studies of other seasonally reproducing primates. For the birth season increase, we propose that the predictable risk of infanticide in this highly seasonal species affects male gonadal and adrenal endocrine activity. We evaluate alternative social and ecological factors influencing the production of both hormone classes and conclude based on our preliminary investigations that none of them can account for the observed pattern

Visual Information Alone Changes Behavior and Physiology during Social Interactions in a Cichlid Fish (Astatotilapia burtoni)

Social behavior can influence physiological systems dramatically yet the sensory cues responsible are not well understood. Behavior of male African cichlid fish, Astatotilapia burtoni, in their natural habitat suggests that visual cues from conspecifics contribute significantly to regulation of social behavior. Using a novel paradigm, we asked whether visual cues alone from a larger conspecific male could influence behavior, reproductive physiology and the physiological stress response of a smaller male. Here we show that just seeing a larger, threatening male through a clear barrier can suppress dominant behavior of a smaller male for up to 7 days. Smaller dominant males being “attacked” visually by larger dominant males through a clear barrier also showed physiological changes for up to 3 days, including up-regulation of reproductive- and stress-related gene expression levels and lowered plasma 11-ketotestesterone concentrations as compared to control animals. The smaller males modified their appearance to match that of non-dominant males when exposed to a larger male but they maintained a physiological phenotype similar to that of a dominant male. After 7 days, reproductive- and stress- related gene expression, circulating hormone levels, and gonad size in the smaller males showed no difference from the control group suggesting that the smaller male habituated to the visual intruder. However, the smaller male continued to display subordinate behaviors and assumed the appearance of a subordinate male for a full week despite his dominant male physiology. These data suggest that seeing a larger male alone can regulate the behavior of a smaller male but that ongoing reproductive inhibition depends on additional sensory cues. Perhaps, while experiencing visual social stressors, the smaller male uses an opportunistic strategy, acting like a subordinate male while maintaining the physiology of a dominant male

Effects of Repeated Petting Sessions on Leukocyte Counts, Intestinal Parasite Prevalence, and Plasma Cortisol Concentration of Dogs Housed in a County Animal Shelter

Objective—To describe changes in WBC counts, plasma cortisol concentration, and fecal parasite shedding of dogs housed in an animal shelter and determine the effects of daily petting sessions on these variables. Design—Hybrid prospective observational and experimental study. Animals—92 healthy dogs newly arrived to an animal shelter and 15 healthy privately owned dogs (control group). Procedures—Blood and fecal samples were collected from shelter dogs 1, 3, and 10 days after arrival and from control dogs once. A subset of shelter dogs (n = 15) was assigned to receive 30 minutes of petting daily. Plasma cortisol concentration was measured, CBCs were performed, and fecal samples were evaluated for parasite ova. Results—For shelter dogs, total leukocyte, neutrophil, and lymphocyte counts increased significantly between days 1 and 10, with less consistent increases in monocyte count and neutrophil-to-lymphocyte count ratio. Parasite shedding was unaffected by duration of shelter stay but was greater for shelter versus control dogs. For shelter dogs, plasma cortisol concentration decreased with time and was higher than that of control dogs on each day. Total leukocyte, neutrophil, and monocyte counts and neutrophil-to-lymphocyte count ratios were also higher for shelter versus control dogs. Petting sessions resulted in a decrease in plasma cortisol concentration but in no other variables. Conclusions and Clinical Relevance—Large increasing immunologic responses, heavy parasite shedding, and high but decreasing plasma cortisol concentration were identified in shelter dogs. Daily 30-minute petting sessions affected only cortisol values, so the clinical importance of petting for immunologic and other health outcomes remains unclear

Effects of Repeated Petting Sessions on Leukocyte Counts, Intestinal Parasite Prevalence, and Plasma Cortisol Concentration of Dogs Housed in a County Animal Shelter

Objective—To describe changes in WBC counts, plasma cortisol concentration, and fecal parasite shedding of dogs housed in an animal shelter and determine the effects of daily petting sessions on these variables. Design—Hybrid prospective observational and experimental study. Animals—92 healthy dogs newly arrived to an animal shelter and 15 healthy privately owned dogs (control group). Procedures—Blood and fecal samples were collected from shelter dogs 1, 3, and 10 days after arrival and from control dogs once. A subset of shelter dogs (n = 15) was assigned to receive 30 minutes of petting daily. Plasma cortisol concentration was measured, CBCs were performed, and fecal samples were evaluated for parasite ova. Results—For shelter dogs, total leukocyte, neutrophil, and lymphocyte counts increased significantly between days 1 and 10, with less consistent increases in monocyte count and neutrophil-to-lymphocyte count ratio. Parasite shedding was unaffected by duration of shelter stay but was greater for shelter versus control dogs. For shelter dogs, plasma cortisol concentration decreased with time and was higher than that of control dogs on each day. Total leukocyte, neutrophil, and monocyte counts and neutrophil-to-lymphocyte count ratios were also higher for shelter versus control dogs. Petting sessions resulted in a decrease in plasma cortisol concentration but in no other variables. Conclusions and Clinical Relevance—Large increasing immunologic responses, heavy parasite shedding, and high but decreasing plasma cortisol concentration were identified in shelter dogs. Daily 30-minute petting sessions affected only cortisol values, so the clinical importance of petting for immunologic and other health outcomes remains unclear

Effects of Repeated Petting Sessions on Leukocyte Counts, Intestinal Parasite Prevalence, and Plasma Cortisol Concentration of Dogs Housed in a County Animal Shelter

Objective—To describe changes in WBC counts, plasma cortisol concentration, and fecal parasite shedding of dogs housed in an animal shelter and determine the effects of daily petting sessions on these variables. Design—Hybrid prospective observational and experimental study. Animals—92 healthy dogs newly arrived to an animal shelter and 15 healthy privately owned dogs (control group). Procedures—Blood and fecal samples were collected from shelter dogs 1, 3, and 10 days after arrival and from control dogs once. A subset of shelter dogs (n = 15) was assigned to receive 30 minutes of petting daily. Plasma cortisol concentration was measured, CBCs were performed, and fecal samples were evaluated for parasite ova. Results—For shelter dogs, total leukocyte, neutrophil, and lymphocyte counts increased significantly between days 1 and 10, with less consistent increases in monocyte count and neutrophil-to-lymphocyte count ratio. Parasite shedding was unaffected by duration of shelter stay but was greater for shelter versus control dogs. For shelter dogs, plasma cortisol concentration decreased with time and was higher than that of control dogs on each day. Total leukocyte, neutrophil, and monocyte counts and neutrophil-to-lymphocyte count ratios were also higher for shelter versus control dogs. Petting sessions resulted in a decrease in plasma cortisol concentration but in no other variables. Conclusions and Clinical Relevance—Large increasing immunologic responses, heavy parasite shedding, and high but decreasing plasma cortisol concentration were identified in shelter dogs. Daily 30-minute petting sessions affected only cortisol values, so the clinical importance of petting for immunologic and other health outcomes remains unclear

Alpha-Melanocyte Stimulating Hormone Reduces Putative Stress-Induced Sickness Behaviors in Isolated Guinea Pig Pups

We have proposed that passive responses observed following maternal separation in guinea pig pups represent “stress-induced sickness behaviors” mediated by proinflammatory processes. In this study, the anti-inflammatory peptide, alpha-melanocyte stimulating hormone (α-MSH) administered intracerebroventricularly, but not intraperitoneally, reduced the passive responses of crouching, eye-closing, and extensive piloerection relative to levels following administration of vehicle. These findings support our hypothesis and are as would be expected if pro-inflammatory processes act centrally to promote the passive behaviors of separated guinea pig pups