Abstract Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Double strand break (DSB) repair mainly occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). We present an assay system that enables simultaneous measurement of all three pathways using Cas9-generated DSBs and next generation sequencing to profile and quantify pathway choice. The assay system has provided several insights. First, absence of the key Alt-EJ factor Pol q only abrogates ~50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. These fundamental differences between BRCA1 and BRCA2 deficiency have implications for therapeutic targeting of HR-deficient cancers. This assay can be used in any system which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Coccidiosis, a parasitic disease of the intestinal tract caused by members of the genera Eimeria and Isospora, is one of the most common and costly diseases in chicken. The aims of this study were to assess the effect of the challenge and level of variability of measured parameters in chickens during the challenge with Eimeria maxima. Furthermore, this study aimed to investigate which parameters are the most relevant indicators of the health status. Finally, the study also aimed to estimate accuracy of prediction for traits that cannot be measured on large scale (such as intestinal lesion score and fecal oocyst count) using parameters that can easily be measured on all animals. The study was performed in 2 parts: a pilot challenge on 240 animals followed by a large-scale challenge on 2,024 animals. In both experiments, animals were challenged with 50,000 Eimeria maxima oocysts at 16 d of age. In the pilot challenge, all animals were measured for BW gain, plasma coloration, hematocrit, and rectal temperature and, in addition, a subset of 48 animals was measured for oocyst count and the intestinal lesion score. All animals from the second challenge were measured for BW gain, plasma coloration, and hematocrit whereas a subset of 184 animals was measured for intestinal lesion score, fecal oocyst count, blood parameters, and plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P < 0.001), oocyst count (P < 0.05), plasma coloration for the optical density values between 450 and 490 nm (P < 0.001), albumin (P < 0.001), α1-globulin (P < 0.01), α2-globulin (P < 0.001), α3-globulin (P < 0.01), and β2-globulin (P < 0.001) were the most strongly affected parameters and expressed the greatest levels of variation. Plasma protein profiles proved to be a new, reliable parameter for measuring response to Eimeria maxima. Prediction of intestinal lesion score and fecal oocyst count using the other parameters measured was not very precise (R2 < 0.7). The study was successfully performed in real raising conditions on a large scale. Finally, we observed a high variability in response to the challenge, suggesting that broilers' response to Eimeria maxima has a strong genetic determinism, which may be improved by genetic selection.

Coccidiosis, a parasitic disease of the intestinal tract caused by members of the genera Eimeria and Isospora, is one of the most common and costly diseases in chicken. The aims of this study were to assess the effect of the challenge and level of variability of measured parameters in chickens during the challenge with Eimeria maxima. Furthermore, this study aimed to investigate which parameters are the most relevant indicators of the health status. Finally, the study also aimed to estimate accuracy of prediction for traits that cannot be measured on large scale (such as intestinal lesion score and fecal oocyst count) using parameters that can easily be measured on all animals. The study was performed in two parts: a pilot challenge on 240 animals, followed by a large-scale challenge on 2,024 animals. In both experiments, animals were challenged with 50,000 Eimeria maxima oocysts at 16 days of age. In the pilot challenge all animals were measured for BW gain, plasma coloration, hematocrit and rectal temperature and in addition a subset of 48 animals was measured for oocyst count and the intestinal lesion score. All animals from the second challenge were measured for BW gain, plasma coloration and hematocrit whereas a subset of 184 animals were measured for intestinal lesion scores, fecal oocyst count, blood parameters, plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P <0.001), oocyst count (P <0.05), plasma coloration for the optical density values between 450 and 490 nm (P <0.001), albumin (P <0.001), $1-globulin (P <0.01), $2-globulin (P <0.001), $3-globulin (P <0.01) and $2-globulin (P <0.001) were the most strongly affected parameters and expressed the greatest levels of variation. Plasma protein profiles proved to be a new, reliable parameter for measuring response to Eimeria maxima. Prediction of intestinal lesion score and fecal oocyst count using the other parameters measured was not very precise (R2 <0.7). The study was successfully performed in real raising conditions on a large scale. Finally, we observed a high variability in response to the challenge, suggesting that broilers’ response to Eimeria maxima has a strong genetic determinism which may be improved by genetic selection.

Chickens from two inbred lines selected for high (L10H) or low (L10L) mannose-binding lectin (MBL) serum concentrations were infected with infectious bronchitis virus (IBV), and innate as well as adaptive immunological parameters were measured throughout the experimental period. Chickens with high MBL serum concentrations were found to have less viral load in the trachea than chickens with low MBL serum concentrations indicating that these chickens were less severely affected by the infection. This study is the first to show that MBL expression is present in the lungs of healthy chickens and that the expression is upregulated at days 3 postinfection (p.i.) in L10H chickens. Furthermore, in the liver of infected chickens, the MBL expression was upregulated at day 7 p.i., despite the fact that the MBL serum concentrations were decreased below baseline at that time point. The number of TCRγδ+CD8α+ cells in the blood of noninfected chickens increased from week 0 to 3 p.i. However, the number of cells was higher in L10H chickens than in L10L chickens throughout the experiment. No increase was observed in the number of TCRγδ+CD8α+ cells in the blood of the infected L10H and L10L chickens. The numbers of B cells at week 3 p.i. were higher for noninfected L10L chickens than for the other chickens. No differences were observed between the infected and noninfected L10H chickens or between the infected L10H and L10L chickens. Furthermore, at week 3 p.i., the number of monocytes was higher in infected and noninfected L10H chickens than in the infected and noninfected L10L chickens. Thus, these results indicate that MBL is produced locally and may be involved in the regulation of the cellular immune response after an IBV infection. However, MBL did not appear to influence the humoral immune response after IBV infection in this study.

For successful genetic dissection of disease resistance it is of great importance to accurately identify the respective phenotypes. In case of coccidiosis some of the conventional phenotypes don’t fully reflect the animal health status. The objective of this study was large-scale evaluation of plasma components as potential parameters for describing health status of broilers challenged with Eimeria maxima. The challenge was performed on 2024 Cobb500 broilers. Plasma coloration was measured on all the animals while measurement of plasma protein content and analysis of plasma protein composition were performed on subset of 184 extreme animals selected based on body weight gain. All fractions of plasma proteins associated with acute phase proteins, except β1-globulin, have been significantly elevated. We observed that the best estimation of plasma coloration variation under a coccidiosis challenge can be obtained by measuring optical density between 450 and 490 nm.

Using genome-wide SNP data, we calculated genomic inbreeding coefficients (FROH  > 1  Mb , FROH  > 2 Mb , FROH  > 8 Mb and FROH  > 16 Mb ) derived from runs of homozygosity (ROH) of different lengths (>1, >2, >8 and > 16 Mb) as well as from levels of homozygosity (FHOM ). We compared these values of inbreeding coefficients with those calculated from pedigrees (FPED ) of 1422 bulls comprising Brown Swiss (304), Fleckvieh (502), Norwegian Red (499) and Tyrol Grey (117) cattle breeds. For all four breeds, population inbreeding levels estimated by the genomic inbreeding coefficients FROH  > 8 Mb and FROH  > 16 Mb were similar to the levels estimated from pedigrees. The lowest values were obtained for Fleckvieh (FPED  = 0.014, FROH  > 8 Mb  = 0.019 and FROH  > 16 Mb  = 0.008); the highest, for Brown Swiss (FPED  = 0.048, FROH  > 8 Mb  = 0.074 and FROH  > 16 Mb  = 0.037). In contrast, inbreeding estimates based on the genomic coefficients FROH  > 1 Mb and FROH  > 2 Mb were considerably higher than pedigree-derived estimates. Standard deviations of genomic inbreeding coefficients were, on average, 1.3-1.7-fold higher than those obtained from pedigrees. Pearson correlations between genomic and pedigree inbreeding coefficients ranged from 0.50 to 0.62 in Norwegian Red (lowest correlations) and from 0.64 to 0.72 in Tyrol Grey (highest correlations). We conclude that the proportion of the genome present in ROH provides a good indication of inbreeding levels and that analysis based on ROH length can indicate the relative amounts of autozygosity due to recent and remote ancestors.

Body: Xylooligosaccharides (XOS) represent a prebiotic candidate demonstrated to increase bifidobacteria in mice feces when supplemented in the diet. As this bifidogenic effect has only been demonstrated in fecal samples, the aim of this study was to investigate if XOS changes the microbiota throughout the entire intestine and examine its effects on mucosal and systemic immune parameters in mice. A 10% XOS supplemented diet revealed a significant increase in the Bifidobacterium group throughout all segments of the intestine with the highest relative increase in ileum. In intestinal epithelial cells (IEC), most genes involved in innate immune responses were unaffected, while the expression of the defensin RegIII (cid:534) , known to limit bacterial colonization of the mucosal surface, was significantly up-regulated in the small intestine. The up-regulation of RegIII (cid:534) may explain the unaffected innate immune response seen in this study, as the homeostatic spatial relationship between the gut microbiota and the host is maintained regardless of the increase in the Bifidobacterium group. We hypothesized that the XOS diet increases SCFA production, and when absorbed, this leads to increased SCFA in the blood. Here, SCFAs bind to the SCFA receptor GPR43 on neutrophils, causing a down-regulation of the pro-inflammatory cytokine IL1 (cid:533) . Indeed, the Il1 (cid:533) and also Ifn (cid:534) expression were significantly decreased by XOS diet, in-dicating that SCFAs modulate the systemic immune response. In vitro treatment of whole blood with propionate led to a decrease in the Il1 (cid:533) expression, thus supporting that increased SCFA production was involved in the Il1 (cid:533) down-regulation observed in the XOS fed mice. A decreased proportion of IFN- (cid:534) producing CD4 T cells were detected in mesenteric lymph nodes of XOS fed mice, but not in spleen. Together with the Il1 (cid:533) and Ifn (cid:534) reduction in blood, this supports an anti-inflammatory effect of XOS diet in both mucosal and systemic immune compartments. Taken together, our results show that XOS feeding decreases systemic and mucosal inflammation and this effect is likely to be due to an increase in bifidobacteria in the small intestine leading to up-regulation of RegIII (cid:534) and increased SCFA production. Abstract Body: Background: Immature dendritic cells (DC) engulf large volumes of extracellular fluid through macropinocytosis. This may be increased further upon ligation of single TLR ligands such as LPS and PAM3CSK4 to TLR on DC (West et al. 2004). In contrast, phagocytosis of bacteria is dependent on specific sequential interactions between the surface of the bacteria and receptors on the DC. How these two different modes of uptake affect the immune response is not well understood. Lactobacillus acidophilus induces a strong IFN- (cid:533) response in bone-marrow derived DC (BMDC) . The induction is dependent on internalization as well as on spleen tyrosine kinase (Syk) signalling (Weiss et al. 2010). Syk is also known to play a role in different types of endocytosis (Y. Tohyama and H.Yamamura 2009). We hypothesise that L. acidophilus uptake via phagocytosis is a prerequisite for IFN- (cid:533) induction. Objective : In the present work the aim is to study the nature of endocytosis involved in uptake of L. acidophilus by DC and how this affects the induction of IFN- (cid:533) . Methods: Endocytosis in DC was quantified by measuring the uptake of fluorescence-labelled dextran or bacteria by flow cytometry and visualised by confocal microscopy. Induction of IFN- (cid:533) and other cytokines in BMDC was measured by QPCR and ELISA. Results: Increased macropinocytosis was induced upon stimulation with the TLR ligands LPS, Pam3CSK4 and the Gram negative E. coli , but not by L. acidophilus . Addition of LPS, Pam3CSK4, or E. coli to the BMDC 30 minutes prior to L. acidophilus increased the uptake of L. acidophilus