Investigating Molecular Mechanisms behind Bacterial Chondronecrosis with Osteomyelitis (BCO) Pathogenesis in Modern Broilers

Bacterial chondronecrosis with osteomyelitis (BCO), a leading cause of lameness in broiler chickens, is characterized by infection, inflammation, and bone attrition. There are currently no effective treatments and positive diagnosis is only possible through necropsy evaluations. Lameness is also a rising animal welfare and economic concern, making prevention and detection of BCO all the more critical. These challenges are exacerbated by a lack of mechanistic understanding of BCO’s etiology. The question I asked during my dissertation was how bacteria induce bone attrition in BCO pathology. My research has shown that mitochondrial dysfunction is characteristic of BCO conditions along with autophagy machinery dysregulation. This autophagy dysregulation is also seen to a result of in vitro infection with known BCO-isolates and affecting bone cell viability. The local bone and systemic blood profile of cytokines, chemokines, inflammasomes, and relevant FGFs were also evaluated. This revealed a unique signature of BCO detectable within circulation and in local bone. Additionally, this signature was made up of factors which negatively affect bone cell viability. It was also shown that primary avian chondrocytes exhibiting optimal phenotypes could be successfully isolated form chicks. These primary cells could provide an improved, highly relevant model for in vitro analysis of avian bone diseases and infections. Finally, the potential roles of two factors regulating energy and lipid metabolism were preliminarily explored as a future target for BCO research. These findings provide novel insight into mechanisms of etiology and means of non-invasive detection while also improving upon current methods of avian growth-plate researc

Effect of Cyclic Heat Stress on Hypothalamic Oxygen Homeostasis and Inflammatory State in the Jungle Fowl and Three Broiler-Based Research Lines

Heat stress (HS) is devastating to poultry production sustainability due its detrimental effects on performance, welfare, meat quality, and profitability. One of the most known negative effects of HS is feed intake depression, which is more pronounced in modern high-performing broilers compared to their ancestor unselected birds, yet the underlying molecular mechanisms are not fully defined. The present study aimed, therefore, to determine the hypothalamic expression of a newly involved pathway, hypoxia/oxygen homeostasis, in heat-stressed broiler-based research lines and jungle fowl. Three populations of broilers (slow growing ACRB developed in 1956, moderate growing 95RB from broilers available in 1995, and modern fast growing MRB from 2015) and unselected Jungle fowl birds were exposed to cyclic heat stress (36 degrees C, 9 h/day for 4 weeks) in a 2 x 4 factorial experimental design. Total RNAs and proteins were extracted from the hypothalamic tissues and the expression of target genes and proteins was determined by real-time quantitative PCR and Western blot, respectively. It has been previously shown that HS increased core body temperature and decreased feed intake in 95RB and MRB, but not in ACRB or JF. HS exposure did not affect the hypothalamic expression of HIF complex, however there was a line effect for HIF-1 alpha (P = 0.02) with higher expression in JF under heat stress. HS significantly up regulated the hypothalamic expression of hemoglobin subunits (HBA1, HBBR, HBE, HBZ), and HJV in ACRB, HBA1 and HJV in 95RB and MRB, and HJV in JF, but it down regulated FPN1 in JF. Additionally, HS altered the hypothalamic expression of oxygen homeostasis- up and down-stream signaling cascades. Phospho-AMPK(Thr172) was activated by HS in JF hypothalamus, but it decreased in that of the broiler-based research lines. Under thermoneutral conditions, p-AMPK(Thr172) was higher in broiler-based research lines compared to JF. Ribosomal protein S6K1, however, was significantly upregulated in 95RB and MRB under both environmental conditions. HS significantly upregulated the hypothalamic expression of NF-kappa B2 in MRB, RelB, and TNF alpha in ACRB, abut it down regulated RelA in 95RB. The regulation of HSPs by HS seems to be family- and line-dependent. HS upregulated the hypothalamic expression of HSP60 in ACRB and 95RB, down regulated HSP90 in JF only, and decreased HSP70 in all studied lines. Taken together, this is the first report showing that HS modulated the hypothalamic expression of hypoxia- and oxygen homeostasis-associated genes as well as their up- and down-stream mediators in chickens, and suggests that hypoxia, thermotolerance, and feed intake are interconnected, which merit further in-depth investigations

Effects of Cyclic Chronic Heat Stress on the Expression of Nutrient Transporters in the Jejunum of Modern Broilers and Their Ancestor Wild Jungle Fowl

snibaThe mechanisms associated between growth rate, gut integrity and heat stress (HS) responses are not known. The current study aimed to evaluate the effect of chronic HS on jejunal nutrient transport in slow- (ACRB from 1950), moderate- (95RAN from 1995), rapid-(modern broilers, MRB) growing birds, and their ancestor wild jungle fowl (JF). One-day male chicks (n=150/line) were placed by line in environmentally controlled chambers and kept under the same environmental conditions until d28. On d29, an 8-h daily cyclic HS (36ºC) was applied to half of the chambers, which lasts until d55, while keeping the rest under thermal neutral conditions (TN, 24°C). Jejunum tissues were collected for morphology assessment and molecular analysis of carbohydrate-, amino acid- and fatty acid- transporters. MRB exhibited the highest BW followed by 95RAN under both conditions. HS decreased FI in MRB and 95RAN, which results in lower BW compared to their TN counterparts, however no effect was observed in ACRB and JF. MRB showed greater villus height to crypt depth ratio under both environmental conditions. Molecular analyses showed that GLUT2, 5, 10, and 11 were upregulated in MRB compared to some of the other populations under TN conditions. HS down regulated GLUT2, 10, 11, and 12 in MRB while it increased the expression of GLUT1, 5, 10, and 11 in JF. GLUT2 protein expression was higher in JF compared to ACRB and MRB under TN conditions. It also showed an increase in ACRB but no effect on 95RAN and MRB under HS conditions. ACRB exhibited greater expression of EAAT3 gene as compared to the rest of populations maintained under TN conditions. HS exposure did not alter the gene expression of amino acid transporters in MRB. Gene expression of CD36 and FABP2 was up-regulated in HS JF birds. Protein expression of CD36 was down-regulated in HS JF while no effect was observed in ACRB, 95RAN and MRB. Taken together, these data are the first to show the effect of HS on jejunal expression of nutrient transporters in three broiler populations known to represent 70 years of genetic progress in the poultry industr

Copy number variants as modifiers of breast cancer risk for BRCA1/BRCA2 pathogenic variant carriers

The risk of germline copy number variants (CNVs) in BRCA1 and BRCA2 pathogenic variant carriers in breast cancer is assessed, with CNVs overlapping SULT1A1 decreasing breast cancer risk in BRCA1 carriers.The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09-1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers.Peer reviewe

Breast and Prostate Cancer Risks for Male BRCA1 and BRCA2 Pathogenic Variant Carriers Using Polygenic Risk Scores

Background: Recent population-based female breast cancer and prostate cancer polygenic risk scores (PRS) have been developed. We assessed the associations of these PRS with breast and prostate cancer risks for male BRCA1 and BRCA2 pathogenic variant carriers. Methods: 483 BRCA1 and 1318 BRCA2 European ancestry male carriers were available from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). A 147-single nucleotide polymorphism (SNP) prostate cancer PRS (PRSPC) and a 313-SNP breast cancer PRS were evaluated. There were 3 versions of the breast cancer PRS, optimized to predict overall (PRSBC), estrogen receptor (ER)-negative (PRSER-), or ER-positive (PRSER+) breast cancer risk. Results: PRSER+ yielded the strongest association with breast cancer risk. The odds ratios (ORs) per PRSER+ standard deviation estimates were 1.40 (95% confidence interval [CI] =1.07 to 1.83) for BRCA1 and 1.33 (95% CI = 1.16 to 1.52) for BRCA2 carriers. PRSPC was associated with prostate cancer risk for BRCA1 (OR = 1.73, 95% CI = 1.28 to 2.33) and BRCA2 (OR = 1.60, 95% CI = 1.34 to 1.91) carriers. The estimated breast cancer odds ratios were larger after adjusting for female relative breast cancer family history. By age 85 years, for BRCA2 carriers, the breast cancer risk varied from 7.7% to 18.4% and prostate cancer risk from 34.1% to 87.6% between the 5th and 95th percentiles of the PRS distributions. Conclusions: Population-based prostate and female breast cancer PRS are associated with a wide range of absolute breast and prostate cancer risks for male BRCA1 and BRCA2 carriers. These findings warrant further investigation aimed at providing personalized cancer risks for male carriers and informing clinical management.Peer reviewe

Polygenic risk scores and breast and epithelial ovarian cancer risks for carriers of BRCA1 and BRCA2 pathogenic variants

Purpose We assessed the associations between population-based polygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks forBRCA1andBRCA2pathogenic variant carriers. Methods Retrospective cohort data on 18,935BRCA1and 12,339BRCA2female pathogenic variant carriers of European ancestry were available. Three versions of a 313 single-nucleotide polymorphism (SNP) BC PRS were evaluated based on whether they predict overall, estrogen receptor (ER)-negative, or ER-positive BC, and two PRS for overall or high-grade serous EOC. Associations were validated in a prospective cohort. Results The ER-negative PRS showed the strongest association with BC risk forBRCA1carriers (hazard ratio [HR] per standard deviation = 1.29 [95% CI 1.25-1.33],P = 3x10(-72)). ForBRCA2, the strongest association was with overall BC PRS (HR = 1.31 [95% CI 1.27-1.36],P = 7x10(-50)). HR estimates decreased significantly with age and there was evidence for differences in associations by predicted variant effects on protein expression. The HR estimates were smaller than general population estimates. The high-grade serous PRS yielded the strongest associations with EOC risk forBRCA1(HR = 1.32 [95% CI 1.25-1.40],P = 3x10(-22)) andBRCA2(HR = 1.44 [95% CI 1.30-1.60],P = 4x10(-12)) carriers. The associations in the prospective cohort were similar. Conclusion Population-based PRS are strongly associated with BC and EOC risks forBRCA1/2carriers and predict substantial absolute risk differences for women at PRS distribution extremes.Peer reviewe

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

Abstract The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.Peer reviewe

Local and Systemic Cytokine, Chemokine, and FGF Profile in Bacterial Chondronecrosis with Osteomyelitis (BCO)-Affected Broilers

Complex disease states, like bacterial chondronecrosis with osteomyelitis (BCO), not only result in physiological symptoms, such as lameness, but also a complex systemic reaction involving immune and growth factor responses. For the modern broiler (meat-type) chickens, BCO is an animal welfare, production, and economic concern involving bacterial infection, inflammation, and bone attrition with a poorly defined etiology. It is, therefore, critical to define the key inflammatory and bone-related factors involved in BCO. In this study, the local bone and systemic blood profile of inflammatory modulators, cytokines, and chemokines was elucidated along with inflammasome and key FGF genes. BCO-affected bone showed increased expression of cytokines IL-1β, while BCO-affected blood expressed upregulated TNFα and IL-12. The chemokine profile revealed increased IL-8 expression in both BCO-affected bone and blood in addition to inflammasome NLRC5 being upregulated in circulation. The key FGF receptor, FGFR1, was significantly downregulated in BCO-affected bone. The exposure of two different bone cell types, hFOB and chicken primary chondrocytes, to plasma from BCO-affected birds, as well as recombinant TNFα, resulted in significantly decreased cell viability. These results demonstrate an expression of proinflammatory and bone-resorptive factors and their potential contribution to BCO etiology through their impact on bone cell viability. This unique profile could be used for improved non-invasive detection of BCO and provides potential targets for treatments

Avian Orexin: Feed Intake Regulator or Something Else?

Originally named for its expression in the posterior hypothalamus in rats and after the Greek word for “appetite”, hypocretin, or orexin, as it is known today, gained notoriety as a neuropeptide regulating feeding behavior, energy homeostasis, and sleep. Orexin has been proven to be involved in both central and peripheral control of neuroendocrine functions, energy balance, and metabolism. Since its discovery, its ability to increase appetite as well as regulate feeding behavior has been widely explored in mammalian food production animals such as cattle, pigs, and sheep. It is also linked to neurological disorders, leading to its intensive investigation in humans regarding narcolepsy, depression, and Alzheimer’s disease. However, in non-mammalian species, research is limited. In the case of avian species, orexin has been shown to have no central effect on feed-intake, however it was found to be involved in muscle energy metabolism and hepatic lipogenesis. This review provides current knowledge and summarizes orexin’s physiological roles in livestock and pinpoints the present lacuna to facilitate further investigations