Layers response to a suboptimal diet through phenotype and transcriptome changes in four tissues

Poultry meat and eggs are major sources of nutrients in the human diet. The long production career of laying hens expose them to biotic or abiotic stressors, lowering their production. Understanding the mechanisms of adaptation to stress is crucial for selecting robust animals and meeting the needs of a growing human population. In this study, financed by the French ChickStress and the European Feed-a-Gene (grant agreement no. 633531) programs, we compared the effects of a 15%-energy-reduced diet (feed stress, FS) vs a commercial diet (control, CT) on phenotypic traits and adipose, blood, hypothalamus and liver transcriptomes in two feed-efficiency-diverging lines. Phenotypic traits showed differences between lines or diets, but no line × diet interaction. In the FS group, feed intake (FI) increased and hens had lower body- and abdominal adipose weight, compared to CT group. We found no differences in egg production or quality. At the transcriptomic level, 16,461 genes were expressed in one or more tissues, 41% of which were shared among tissues. We found differentially expressed genes between lines or diet in all tissues, and almost no line × diet interactions. Focusing on diet, adipose and liver transcriptomes were unaffected. In blood, pathways linked to amino acids, monosaccharides, and steroid metabolism were affected, while in the hypothalamus, changes were observed in fatty acid metabolism and endocannabinoid signalling. Given the similarities in egg production, the FS animals seem to have adapted to the stress by increasing FI and by mobilizing adipose reserves. Increase in FI did not appear to affect liver metabolism, and the mobilization of adipose reserves was apparently not driven at the transcriptomic level. In blood, the pathways linked to metabolic processes suggest a metabolic role for this tissue in chicken, whose erythrocytes are nucleated and contain mitochondria. FI increase might be linked to the hypothalamic pathway of endocannabinoid signalling, which are lipid-based neurotransmitters, notably involved in the regulation of appetite

Pitfalls in machine learning‐based assessment of tumor‐infiltrating lymphocytes in breast cancer: a report of the international immuno‐oncology biomarker working group

The clinical significance of the tumor-immune interaction in breast cancer (BC) has been well established, and tumor-infiltrating lymphocytes (TILs) have emerged as a predictive and prognostic biomarker for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2 negative) breast cancer (TNBC) and HER2-positive breast cancer. How computational assessment of TILs can complement manual TIL-assessment in trial- and daily practices is currently debated and still unclear. Recent efforts to use machine learning (ML) for the automated evaluation of TILs show promising results. We review state-of-the-art approaches and identify pitfalls and challenges by studying the root cause of ML discordances in comparison to manual TILs quantification. We categorize our findings into four main topics; (i) technical slide issues, (ii) ML and image analysis aspects, (iii) data challenges, and (iv) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns, or design choices in the computational implementation. To aid the adoption of ML in TILs assessment, we provide an in-depth discussion of ML and image analysis including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial- and routine clinical management of patients with TNBC

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.Peer reviewe

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Peer reviewe

Refined histopathological predictors of BRCA1 and BRCA2 mutation status : a large-scale analysis of breast cancer characteristics from the BCAC, CIMBA, and ENIGMA consortia

Abstract Introduction The distribution of histopathological features of invasive breast tumors in BRCA1 or BRCA2 germline mutation carriers differs from that of individuals with no known mutation. Histopathological features thus have utility for mutation prediction, including statistical modeling to assess pathogenicity of BRCA1 or BRCA2 variants of uncertain clinical significance. We analyzed large pathology datasets accrued by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and the Breast Cancer Association Consortium (BCAC) to reassess histopathological predictors of BRCA1 and BRCA2 mutation status, and provide robust likelihood ratio (LR) estimates for statistical modeling. Methods Selection criteria for study/center inclusion were estrogen receptor (ER) status or grade data available for invasive breast cancer diagnosed younger than 70 years. The dataset included 4,477 BRCA1 mutation carriers, 2,565 BRCA2 mutation carriers, and 47,565 BCAC breast cancer cases. Country-stratified estimates of the likelihood of mutation status by histopathological markers were derived using a Mantel-Haenszel approach. Results ER-positive phenotype negatively predicted BRCA1 mutation status, irrespective of grade (LRs from 0.08 to 0.90). ER-negative grade 3 histopathology was more predictive of positive BRCA1 mutation status in women 50 years or older (LR = 4.13 (3.70 to 4.62)) versus younger than 50 years (LR = 3.16 (2.96 to 3.37)). For BRCA2, ER-positive grade 3 phenotype modestly predicted positive mutation status irrespective of age (LR = 1.7-fold), whereas ER-negative grade 3 features modestly predicted positive mutation status at 50 years or older (LR = 1.54 (1.27 to 1.88)). Triple-negative tumor status was highly predictive of BRCA1 mutation status for women younger than 50 years (LR = 3.73 (3.43 to 4.05)) and 50 years or older (LR = 4.41 (3.86 to 5.04)), and modestly predictive of positive BRCA2 mutation status in women 50 years or older (LR = 1.79 (1.42 to 2.24)). Conclusions These results refine likelihood-ratio estimates for predicting BRCA1 and BRCA2 mutation status by using commonly measured histopathological features. Age at diagnosis is an important variable for most analyses, and grade is more informative than ER status for BRCA2 mutation carrier prediction. The estimates will improve BRCA1 and BRCA2 variant classification and inform patient mutation testing and clinical management

Phenotype and multi-tissue transcriptome response to diet changes in laying hens

Adaptation to feed changes in laying hens is particularly important to promote innovation, in selection schemes and in dietary solutions, for the sustainability of the egg-production sector. In Europe and USA poultry feed is rich in cereals, while Asian countries privilege by-products to soybean meal, resulting in low energy diets.In this study, we investigated the effects of a sub-optimal low energy diet on different traits and multi-tissue transcriptomes of brown egg layers of 2 divergent lines selected for low (R-) and high (R+) residual feed intake. The 2 diets had a similar protein content, while the energy content was reduced by 15% as compared to the standard diet (2450 Kcal versus 2800 Kcal). The R+ and R- hens were fed ad libitum, with the standard diet until 17 wk of age; half of the birds were fed with the low energy feed until 33 wk of age when a subset of birds was slaughtered for tissue sampling (8 per line and diet). Food intake was increased in response to the suboptimal diet whereas egg number was unchanged showing that birds were able to adjust their energy intake by modifying feed intake. Nevertheless, hens fed the low energy diet had a higher feed efficiency (pval < 5%) and a higher residual feed intake (P-value <1%). No diet × line interaction was observed for these traits. PolyA+ RNA from different tissues were sequenced resulting in 90 M reads per sample. After bioinformatics treatment and differential analysis, we observed in liver and adipose tissue only few differentiallyexpressed genes (DEG) between diets (16 and 21 respectively). In contrast, we observed in blood 1179 DEG out of 17123 expressed genes with 463 and 716 over and under expressed in the suboptimal diet compared to the standard diet. No diet × line interaction was observed in the three tissues. GObp term enrichment revealed that under expressed DEG in blood were associated with glucose catabolism, cholesterol biosynthesis, mitotic cell cycle and protein catabolic process. Taken together, these results indicate an adaptation of birds to diet changes by increasing feed intake to maintain egg production, and a tissue-specific response with a limited role of metabolic tissues as liver and adipose tissue compared to the blood

Layers response to suboptimal diet through phenotypic and transcriptomic changes in four tissues

International audiencePoultry meat and eggs are major sources of nutrients in human alimentation. The long production career of layers expose them to numerous stressors. Understanding the adaptive mechanisms is crucial to select robust animals and meet the needs of a growing human population. In this context, we compared the effects of a 15%-energy-reduced diet (LE) vs. a commercial diet (CT) in two feed-efficiency-diverging chicken lines. We first studied the effects of the LE on performances (egg mass, feed intake [FI], body weight) and body energy reserves, before investigating the molecular mechanisms at work by studying the adipose tissue, blood, hypothalamus and liver transcriptomes. The absence of differences in egg production between diets, combined to the increase in feed intake and the decrease in body weight in LE suggest an adaptation to the diet through an increase in energy input and a mobilization of body energy reserves. The birds reacted similarly to the diet in both lines, as shown by the absence of significant line×diet interactions. At the transcriptomic level, no interactions were detected. The increase in FI did not affect the liver, neither did the mobilization of body reserves for the adipose tissue. In blood, the differentially expressed genes were related to amino-acids, monosaccharides and steroid metabolisms. In the hypothalamus, the fatty acids metabolism and endocannabinoid signaling pathways were affected. We propose a mechanism involving endocannabinoids that could explain the increase in FI and therefore part of the adaptation to the low-energy diet. Work funded by ANR-13-ADAP-0014 and H2020-Feed-a-Gene projects

Liver and whole blood transcriptome response to chronic heat exposure in laying hens

Adaptation to heat exposure is required to maintain animal welfare and productivity under high ambient temperature (AT) conditions. In this study we investigate the effects of chronic heat exposure (5 weeks at a constant temperature of 32°C) on the liver and whole blood transcriptome of brown egg layers from 2 divergent lines selected for low (R-) and high (R+) residual feed intake. The R+ and R- hens were equally distributed among 2 temperature-controlled chambers and reared under thermo-neutrality (22°C). At 28 wk of age the AT of one chamber was increased to 32°C until 33 wk of age, when 32 animals (8 per line and treatment) were slaughtered. Total RNA was obtained from the liver and blood and was sequenced using the Illumina HiSEqn 3000, yielding an average per sample of 90 million paired-end reads. The reads were mapped to the Gallus gallus-5 reference genome by STAR software and counted by RSEM software using the Ensembl V87 GTF annotation. Comparisons between the two AT groups were made using the edgeR-robust R/Bioconductor package. Patterns of AT-specific differential expression were largely shared by the two lines, and no evidence of temperature × line interactions were observed. In liver, a total of 229 differentially expressed genes (DEG) were identified (adjusted P-values < 0.05) with respectively 104 and 125 over and under expressed in the heat-exposed compared to the control group. In blood, 960 DEG were identified between the two AT groups with 479 and 481 over and under expressed. Most DEG were tissue specific, and only 18 genes were DE in both liver and blood. Ingenuity Pathway Analysis revealed that many of the DEG in liver were associated with amino acid and lipid metabolisms and energy production. Key genes involved in fatty acid β-oxidation, ketogenesis, cholesterol biosynthesis were under-expressed in the heat-exposed animals. In blood, many of the DEG were associated with cell related functions. Based on the DEG expression profile, down-regulation was observed for the PI3K/AKT, the VEGF and the PDGF signalling pathways involved in cell survival and growth, vasculogenesis and angiogenesis. Taken together, these results indicate a tissue-specific response to heat exposure

Chicken adaptive response to low energy diet: main role of the hypothalamic lipid metabolism revealed by a phenotypic and multi-tissue transcriptomic approach

International audienceBackground: Production conditions of layer chicken can vary in terms of temperature or diet energy content compared to the controlled environment where pure-bred selection is undertaken. The aim of this study was to better understand the long-term effects of a 15%-energy depleted diet on egg-production, energy homeostasis and metabolism via a multi-tissue transcriptomic analysis. Study was designed to compare effects of the nutritional intervention in two layer chicken lines divergently selected for residual feed intake. Results: Chicken adapted to the diet in terms of production by significantly increasing their feed intake and decreasing their body weight and body fat composition, while their egg production was unchanged. No significant interaction was observed between diet and line for the production traits. The low energy diet had no effect on adipose tissue and liver transcriptomes. By contrast, the nutritional challenge affected the blood transcriptome and, more severely, the hypothalamus transcriptome which displayed 2700 differentially expressed genes. In this tissue, the low-energy diet lead to an over-expression of genes related to endocannabinoid signaling (CN1R, NAPE-PLD) and to the complement system, a part of the immune system, both known to regulate feed intake. Both mechanisms are associated to genes related polyunsaturated fatty acids synthesis (FADS1, ELOVL5 and FADS2), like the arachidonic acid, a precursor of anandamide, a key endocannabinoid, and of prostaglandins, that mediate the regulatory effects of the complement system. A possible regulatory role of NR1H3 (alias LXRα) has been associated to these transcriptional changes. The low-energy diet further affected brain plasticity-related genes involved in the cholesterol synthesis and in the synaptic activity, revealing a link between nutrition and brain plasticity. It upregulated genes related to protein synthesis, mitochondrial oxidative phosphorylation and fatty acid oxidation in the hypothalamus, suggesting reorganization in nutrient utilization and biological synthesis in this brain area. Conclusions: We observed a complex transcriptome modulation in the hypothalamus of chicken in response to low-energy diet suggesting numerous changes in synaptic plasticity, endocannabinoid regulation, neurotransmission, lipid metabolism, mitochondrial activity and protein synthesis. This global transcriptomic reprogramming could explain the adaptive behavioral response (i.e. increase of feed intake) of the animals to the low-energy content of the diet

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study

International audienceAbstract Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error ( p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections ( p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor