This work using an ex vivo osteochondral mechanically stimulated advanced platform demonstrated that recurrent mechanical stress at early time points impeded cell migration into the hydrogel, providing a unique opportunity to improve our understanding on management of joint injury.Cell migration has a central role in osteochondral defect repair initiation and biomaterial-mediated regeneration. New advancements to reestablish tissue function include biomaterials and factors promoting cell recruitment, differentiation and tissue integration, but little is known about responses to mechanical stimuli. In the present pilot study, we tested the influence of extrinsic forces in combination with biomaterials releasing chemoattractant signals on cell migration. We used an ex vivo mechanically stimulated osteochondral defect explant filled with fibrin/hyaluronan hydrogel, in presence or absence of platelet-derived growth factor-BB or stromal cell-derived factor 1, to assess endogenous cell recruitment into the wound site. Periodic mechanical stress at early time point negatively influenced cell infiltration compared to unloaded samples, and the implementation of chemokines to increase cell migration was not efficient to overcome this negative effect. The gene expression at 15 days of culture indicated a marked downregulation of matrix metalloproteinase  Articular cartilage plays a key role in the function of joints, and when damaged it becomes inefficient to withstand harsh conditions over time, posing a significant challenge among clinicians. The very poor intrinsic healing capacity of this tissue in combination with the high incidence of trauma place at risk many asymptomatic young and healthy patients toward the evolution of degenerative conditions with reduced possibility of interventions . Surgical procedures including microfracture and osteochondral allografts are being applied in clinical practice. While the former is far from being successful in replacing the damaged cartilage by repair tissue with long-lasting hyaline properties , the latIn recent years the modulation of biochemical and biophysical cues, when considering the design of biomaterials used as 3D templates for tissue regeneration, has advanced our understanding of cartilage repair processes . These dIn agreement with a body of evidence from literature, our previous work on cartilage healing using in vitro studies and a model for osteochondral defect repair after subcutaneous implantation in mice suggest that stiffer materials represent a barrier to endogenous healing , where mThe success of material-based systems for osteochondral defect repair depends on the ability of the scaffolds to sustain compressive, shear and tensile forces during joint loading. Although most hydrogels are not ideal materials to resist complex motion, mechanical properties can be enhanced by modifying polymers with functional groups to form hydrophilic structures and increase the crosslinking density in the network . Fibrin/2. The medium, referred to as chondro-permissive medium, was changed three times per week.Osteochondral explants were harvested from stifle joints of five to eight-months-old calves, obtained from a local abattoir  within 48 h of slaughter. Previous studies using the same timeframe have shown explant viability preservation for up to 28 days ,37. Cyliw/v ratio of 3.2:1) were used with HA molecular weight of 235 kDa . Briefly, HA was first reacted with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide  and N-hydroxysuccinimide  to convert part of its carboxylic groups to NHS-active ester moieties. In a second step, a buffered solution of fibrinogen  was reacted with the HA active ester solution to produce a clear FB/HA conjugate solution. Hydrogels were then prepared by mixing thrombin solution  containing calcium chloride (1M CaCl2) with FB/HA conjugate and polymerizing at 37 °C for 30 min. The rheological features of the resulting hydrogels were characterized in previous works [FB/HA conjugates were synthesized via a two-step reaction as previously described . Final cus works ,38.PDGF-BB or SDF-1α  were added to the FB/HA conjugate solution prior polymerization to obtain final concentrations of 2 µg/mL of PDGF-BB or 10 µg/mL of SDF-1α, respectively. The selected dose of PDGF-BB used in the following experiments was chosen based on our previous FB/HA hydrogel release study , since t2 incubator at 37 °C, 5% CO2, 85% humidity [For ex vivo explant culture, 50 µL of FB/HA or FB/HA carrying chemotactic factors were cast into the osteochondral explants after defect creation. Then, osteochondral explant constructs were cultured in 3 mL of chondro-permissive medium and loaded in our bioreactor system. Osteochondral plugs underwent mechanical stimulation using a four-station bioreactor system, installed in a COhumidity . A ceramOne hour of mechanical loading was performed per day over either 6 days or 15 days from the start of the culture .Samples for histology were fixed in 4% buffered formaldehyde  for 24 h, dehydrated until absolute ethanol, then embedded in methyl methacrylate (MMA) and sectioned in 130 µm sections. For staining, slides were treated with 1% formic acid and subsequently rinsed in tap water and dHn = 3/group) were divided in three subsections of 1 mm height . Cell colonization into the defect was assessed at day 15 by counting cell infiltration number in the defect area following specific criteria. Osteochondral defects of Toluidine blue stained cross-sections (® probes for collagens type-I (COL1A2), type-II (COL2A1), aggrecan (ACAN), matrix metalloproteinase 3 (MMP-3), MMP-13, and the catalogue numbers of the gene expression assays used for amplification of ribosomal protein lateral stalk subunit P0 (RPLP0), versican (VCAN), β1-integrin (TFB1M), and beta-actin (ACTB) . Data collected at day 15 were expressed as relative values of target mRNA and determined according to the comparative CT method. First the target gene expression was normalized to the expression of the reference gene RPLP0. This reference gene had been shown to remain stable under mechanical loading conditions, whereas other commonly used reference genes such as glyceraldehyde 3-phosphate dehydrogenase GAPDH may be affected by mechanical load [After 15 days of culture, FB/HA hydrogels were removed from the explant, homogenized using the Tissue Lyser system , and total RNA of the migrated cells was extracted using AllPrep DNA/RNA Micro Kit (Qiagen). RNA concentration and quality were measured using NanoDrop 1000 spectrophotometer . cDNA was prepared using SuperScript Vilo IV Master Mix (ThermoFisher) according to the manufacturer’s instructions and real time PCR was performed on a Quant Studio Flex 6 instrument (ThermoFisher). cal load . In a seHydrogels were assessed for DNA content after removing the FB/HA hydrogel from the osteochondral explants followed by homogenization in a Tissue Lyzer for sample disruption . DNA was purified using AllPrep DNA/RNA Micro Kit (Qiagen), and its content measured by Qubit 1X dsDNA HS assay kit following manufacturer’s instruction .p < 0.05.Data were analyzed by using SPSS software, and the results are expressed as mean ± standard deviation (SD). Two independent experiments were performed using triplicates per group for early cell migration studies at day 6 and 15. Due to the non-symmetrical data distribution, a non-parametric test was selected to analyze the DNA content and the gene expression data. DNA amounts of samples treated with chemoattractant or mechanical load were expressed relative to the DNA content of untreated control samples from the same bovine donor to normalize for donor variation in basal cell migration. Similarly, gene expression data of samples treated with chemoattractant or mechanical load were expressed relative to the levels of untreated control samples. Independent samples were then statistically assessed by Kruskal–Wallis test and pairwise comparisons. For quantification of cells migrated into the osteochondral samples after 15 days of culture, three explants per group and three sections per sample were used; statistically significant differences between unloaded and loaded groups were determined by Kruskal–Wallis test and pairwise comparisons. Statistical significance was considered for To determine the effect of loading on defect colonization and evaluate PDGF-BB and SDF-1α as efficient chemotactic factors for cells present in the ex vivo osteochondral explants, migrated cells were assessed as function of mechanical stress and chemoattractant delivery. To achieve that, osteochondral defect plugs filled with FB/HA hydrogel in presence or absence of 2 µg/mL PDGF-BB or 10 µg/mL SDF-1α were cultured for 6 and 15 days with or without exposure to mechanical stimuli. Toluidine blue staining revealed that endogenous cells interacted with FB/HA hydrogel; cells started adhering and infiltrating the defect within 15 days, while no or very few cells were visible at 6 days B. MechanTo quantitatively assess the invasion of endogenous cells into the hydrogel delivered to the osteochondral defect explants, DNA measurement and cell counting were performed. DNA content analysis suggested that the exposure to mechanical stimuli tended to decrease cell recruitment at day 6 and day 15. Although a slight increase in DNA was found in the loaded compared to the unloaded control samples, these differences were not statistically significant C,D. The p < 0.05; p < 0.05, The cell colonization along the osteochondral explant depth at day 15 was further evaluated by histology. Sagittal sections of explants were cut to permit cell counting in order to explore endogenous cell migration in the entire defect A and in MMP13 in loaded constructs without or with SDF-1α compared to their respective unloaded samples  , appearsQuantitative DNA measurements did not show statistically significative differences at day 6 and day 15, even though fewer cells seemed to populate FB/HA hydrogels exposed to complex load. In support of our observations, total cell count indicated that complex articulating motion significantly decreased cell invasion in loaded control plugs compared to unloaded controls. These results suggested that mechanical stimuli negatively influenced cell migration by slowing down this process at early time points. We can, however, not exclude that this effect could be due to an inhibition of cell proliferation or an enhanced cell death .It is important to mention that the SDF-1α and PDGF-BB gradients did not significatively enhance migration in our ex vivo model. Our previous in vivo study on osteochondral repair showed that the exposure of osteochondral defect explants with FB/HA hydrogels to 1 µg/mL PDGF-BB before implantation did not significantly enhance cell recruitment compared to untreated constructs . AlthougThe interface layer (S2) showed the highest cell invasion in unloaded control constructs in comparison to the adjacent layers, suggesting a new potential pattern of migration in the osteochondral unit where either cells present in the subchondral bone or in the calcified cartilage highly participate in defect restoration ,50,51. PMMP13 and MMP3 gene expression were no longer present upon the addition of the chemokines. The joint motion simulator did not affect the mRNA ratio of Collagen II to Collagen I and Aggrecan to Versican, indicating minimal influence on the chondrocytic phenotype [The loading protocol was chosen based on previous protocols tested by our group. Antunes et al. investigated the effect of low intensity motion set-ups and a bioactive agent on gene expression of primary bovine chondrocytes seeded FB/HA hydrogels; due to the low resilience of the hydrogel, samples were subjected to an offset displacement of 10% and low amplitude dynamic axial compression between 10% and 11.5% . Convershenotype . Our celβ1 integrin and upregulating actin expression. Since integrins provide the main molecular link attaching cells to extracellular matrix, and the bonds that link actin cytoskeleton to integrins dynamically break and reform [Periodic mechanical stress may induce a reduction of endogenous cells adhesion in the defect at early time points by downregulating the expression of d reform , it is pAlthough any building blocks need physical forces in order to assemble and hold themselves together , we cannTaken together these data suggest that the applied mechanical stimuli did not enhance cell recruitment into the osteochondral defect at early time point and the provided chemotactic agents did not influence this process. This might indicate that a well-orchestrated mechanical loading over time is crucial for successful design of endogenous cell recruitment and cartilage healing studies. After an observation period of 15 days, cell infiltration was evident, while the number of migrated cells was still limited. Due to this limitation, no extended evaluation of cell types, matrix synthetic activity and matrix composition could be performed besides the gene expression analysis. A parameter that needs more rigorous attention is the pre-culture time of the osteochondral hydrogel constructs, as it can play a pivotal role for tissue maturation and integration . Indeed,The present short report details the temporal and spatial migration pattern in a mechanically stimulated ex vivo osteochondral defect explant filled with FB/HA hydrogel, demonstrating that loading post defect creation might inhibit the endogenous cell migration potential. The implementation of chemokines to increase cell migration was not efficient to overcome this negative effect. This study highlights a significant improvement in the understanding of osteochondral wound healing, suggesting that well-orchestrated mechanical application over time could be the prelude for enhancing cell mobilization and differentiation. The model is useful to decode the interplay between cells, hydrogel, mechanical and biochemical factors; it may unravel the dynamic process of endogenous cell recruitment and signaling pathways implicated in the repair. In light of the inherent advantages that could be utilized based on the modulation of different stimuli, the model represents an attractive system to improve our understanding about the management of joint injury and rehabilitation protocols. Longer-term studies will be required to assess hydrogel-guided neo-cartilage formation and neo tissue integration.


Rationale: Idiopathic and heritable pulmonary arterial hypertension (PAH) are rare but comprise a genetically heterogeneous patient group. RNA sequencing linked to the underlying genetic architecture can be used to better understand the underlying pathology by identifying key signaling pathways and stratify patients more robustly according to clinical risk.Objectives: To use a three-stage design of RNA discovery, RNA validation and model construction, and model validation to define a set of PAH-associated RNAs and a single summarizing RNA model score. To define genes most likely to be involved in disease development, we performed Mendelian randomization (MR) analysis.Methods: RNA sequencing was performed on whole-blood samples from 359 patients with idiopathic, heritable, and drug-induced PAH and 72 age- and sex-matched healthy volunteers. The score was evaluated against disease severity markers including survival analysis using all-cause mortality from diagnosis. MR used known expression quantitative trait loci and summary statistics from a PAH genome-wide association study.Measurements and Main Results: We identified 507 genes with differential RNA expression in patients with PAH compared with control subjects. A model of 25 RNAs distinguished PAH with 87% accuracy  in model validation. The RNA model score was associated with disease severity and long-term survival (P = 4.66 × 10−6) in PAH. MR detected an association between SMAD5 levels and PAH disease susceptibility .Conclusions: A whole-blood RNA signature of PAH, which includes RNAs relevant to disease pathogenesis, associates with disease severity and identifies patients with poor clinical outcomes. Genetic variants associated with lower SMAD5 expression may increase susceptibility to PAH. Blood transcriptomic profiles in pulmonary arterial hypertension have been examined in small limited studies indicating their potential utility in differentiating patients from control subjects and by clinical phenotypes.This study represents the most comprehensive analysis of whole-blood RNA profiles in pulmonary arterial hypertension and identifies and validates a signature that both differentiates patients from control subjects and stratifies patients by clinical risk and severity. Genetic analyses (Mendelian randomization) indicate potential causal roles for some genes including the TGF-β signaling molecule SMAD5.Pulmonary arterial hypertension (PAH) is associated with vasoconstriction and occlusion of distal pulmonary arteries, characterized by endothelial damage, smooth muscle and fibroblast proliferation, and inflammation. Increased pulmonary vascular resistance leads to right heart failure, with survival rates estimated at 52–75% at 5 years, even with modern-day therapy . The ratTranscriptome profiling through RNA sequencing permits a comprehensive analysis of gene expression in tissue samples. Whole-blood RNA analysis offers an alternative “liquid biopsy” to lung biopsy, which carries a high risk in PAH, and can be performed sequentially. This approach can also investigate immune mechanisms in PAH that have recently been highlighted . PreviouThe aim of this study was to characterize gene pathways associated with PAH and to assess their association with disease heterogeneity, specifically in terms of disease severity and outcomes including response to vasodilators and mortality, and genetic background. We compare gene expression in whole-blood samples from 359 patients with idiopathic, heritable, or drug-induced PAH from the UK PAH Cohort study with 72 age- and sex-matched healthy volunteers without any cardiac or respiratory disease as control subjects. Using equal distribution of samples into a three-stage design, we identified reproducible RNA expression differences by RNAseq. Two distinct computational approaches were used to estimate white blood cell (WBC) fractions and thus account for the potential effect of different cell numbers on gene transcript levels across samples. A predictive statistical model that combined gene expression differences performed well at identifying patients with PAH in a separate case–control analysis. The RNA-based score was also associated with disease severity and clinical outcomes . Enrichment of genetic variants that determine the levels of PAH RNAs was detected, implicating these RNAs in the pathogenesis of the disease.Comprehensive methods are included in the online supplement.www.ipahcohort.com). In each case, diagnosis was confirmed by right heart catheterization following established international guidelines (see online supplement for further details). Genomics data were obtained from a published PAH genome-wide association study  were recruited from expert centers across the UK as part of the PAH Cohort study , RNA validation (n = 120), and model validation (n = 119). Each of these three groups was then compared with an independent set of age- and sex-matched healthy volunteers as control subjects  were analyzed using Salmon v0.9.1 . Differential expression analysis was performed using edgeR v3.22.5 (P < 0.05) directionally consistent in the initial analyses and meeting false discovery rate (FDR) multiple test corrections  in the combined analysis were taken forward. Five hundred seven genes meeting these criteria were considered to generate a model to distinguish PAH from control subjects. Subset selection of RNAs that best distinguish PAH in combination was performed by least absolute shrinkage and selection operator (LASSO) regression analysis, using the glmnet v2.0-18 package from CRAN, with k-fold cross-validation (k = 10) selecting the largest value of lambda such that error is within 1 SE of the minimum. This produces an RNA score from a linear weighted combination of the mRNAs identified by the LASSO analysis. Receiver operating characteristic analysis was performed using the pROC v1.14.0 package from Bioconductor  and World Health Organization (WHO) functional class  and by cardiac biomarkers .david.ncifcrf.gov) and Ingenuity Pathway Analysis (IPA) using in-built FDR corrections for multiple tests.Functional annotation and enrichment of the genes associated with PAH was performed using DAVID (Mendelian randomization (MR) analysis using all independent genome-wide significant whole-blood expression quantitative trait loci (eQTL) from two published studies , 16 and P < 0.05) between PAH and control subjects with directional concordance in both discovery and validation analyses . All 507 genes were included after accounting for multiple testing . None were associated with exposure to the main PAH therapies (see online supplement for further details). These included RNAs in pathways relevant to PAH; for example, SMAD5 (mothers against decapentaplegic homolog-5), encoding a downstream mediator of signaling of BMPR2 . All real-time quantitative PCR measurements correlated significantly with RNAseq quantification .A set of differentially expressed RNAs relevant to PAH pathobiology were selected for validation of RNAseq quantification by real-time quantitative PCR, namely, n = 119 patients with PAH and n = 24 control subjects) and demonstrated an area under the curve of 0.868  . The model was then tested in an independent validation set (1–0.945) . The optWe next examined whether the RNA model was also associated with patients with the poorest outcomes, using all 359 patients. The optimum cutoff (1.910) for identifying PAH nonsurvivors with the LASSO model separated patients with PAH into low- and high-risk groups in survival analysis  and E3.To determine which of the 25 transcripts in the model were responsible for the association with survival, we tested each transcript for associations with all-cause mortality during follow-up. After FDR correction for multiple testing, three intronic long noncoding RNAs  were associated with survival in all the patients with PAH analyzed and cutoffs distinguished high- and low-risk patient subgroups .P = 0.008; P = 5.10 × 10−4; P = 8.7 × 10−5).To further characterize the RNA signature, we analyzed its association with three clinical measures of disease severity—WHO functional class, exercise capacity , and cardiac biomarkers (BNP or NT-proBNP). We found a significant difference in RNA scores between patients in different WHO functional classes  was consistently lower in PAH.Three hundred seventy-two out of the 507 top dysregulated genes from the current RNAseq study were present in the lung tissue microarray study and 161/372 (43%) were also directionally consistent. Forty-one out of 161 (25%) genes were nominally significant and 26 met FDR corrected significance (Table E9). Only one gene was found to be dysregulated in patients with PAH across all three studies: AMD1, the only gene in common between the current RNAseq study, the recent lung tissue microarray study and the gene expression meta-analysis, was part of the top IPA gene network .Of the 507 RNAs found to be differentially expressed in PAH, 435 were present in the functional annotations in DAVID. Enrichment of DNA-binding TFs (transcription factors), such as HIF1α (hypoxia-inducible factor 1α) and KLF10 (Krüppel-like factor 10), and many zinc finger–containing TFs was observed compared with a background of the genes detected (Table E10). The Ingenuity Knowledge Base mapped 505/507 transcripts. Double-stranded DNA repair, T-cell receptor, PI3K signaling in B lymphocytes, the role of JAK family kinases in IL6-type cytokine signaling and hypoxia signaling were among the top canonical pathways identified by IPA . SESN1 (Sestrin-1) and SMAD5, reached nominal significance using eQTL in both data sets  was associated with an 8.5% reduction in the risk of developing PAH . SMAD5 levels were similarly reduced in patients with PAH with and without pathogenic BMPR2 variants , supporting the observation that impaired signaling in the BMPR2 pathway is more common in PAH than rare mutations in BMPR2 suggest. The PAH RNA model score was similarly elevated in patients with PAH with and without pathogenic BMPR2 variants .The SMAD5 are more common in patients with PAH.Here we report an RNA signature that separates idiopathic and heritable PAH from healthy individuals. The signature also stratifies patients according to disease severity and risk of early death, adding plausibility to its association with PAH. Several of the discriminating mRNAs encode TFs, including SMAD5, HIF-1α and KLF10. MR analysis to integrate genomic data and identify underlying pathogenic signaling pathways revealed that genetic variants associated with lower expression levels of SMAD5 encodes an intracellular transcriptional modulator that is activated by ligand binding of BMPR2, the most common genetic risk factor in heritable PAH (MR is a powerful tool for separating cause from consequence, as an individual’s genetic status predates the development of PAH. We harnessed genetic data from a published international genome-wide association study  and infoable PAH . SMAD5 aable PAH . SMAD5 aable PAH , which mable PAH . Novel table PAH –25. WholAMD1 was the only gene consistently dysregulated in both these studies and our data set, with lower levels in PAH samples in all three studies. AMD1 encodes a key enzyme controlling the supply of decarboxylated S-adenosylmethionine for polyamine biosynthesis and is regulated by several mechanisms, including increased protein degradation in the presence of elevated polyamines and the inhibition of mRNA translation by spermidine and spermine (AMD1 expression in patients with PAH may be in part due to negative feedback from elevated polyamines. These data contrast with observations in hypoxic rodents. Amd1 expression is increased in hypoxic animals, and both AMD1+/− mice and mice treated with the AMD1 inhibitor, SAM486a, were partially protected from the development of hypoxic PH (ATP13A3 with PAH (We were able to externally validate our findings with a meta-analysis of published PAH blood transcriptome studies  and a PAspermine . We havespermine . Reducedpoxic PH . Interespoxic PH . This miwith PAH .Many of the transcripts that passed robust statistical evaluation in this study are novel. We identified three specific long noncoding RNAs that are reduced in PAH and associated with poor outcomes. These transcripts are not well studied, and their role in regulating lysosomal proton pump protein ATP6V0E2, protein tyrosine phosphatase PTP4A2, or small nuclear ribonucleoprotein-associated protein N remain to be established. Their association with survival selects these out as worthy of further investigation. In addition to suggesting biological relevance, the association of the RNA model, developed to distinguish PAH from control subjects, with survival and disease severity in patients with PAH suggests that the differences observed could be useful to identify patients who may require a more aggressive treatment strategy, such as upfront triple therapy . This stThere is considerable interest in developing a biochemical test to identify patients with PAH who respond well to calcium antagonists that would supersede the current test, namely, an acute vasodilator challenge while undergoing cardiac catheterization. In contrast to previous reports, we were unable to demonstrate the association of a peripheral transcript signature to vasoresponder status, including previously studied RNAs . A stren+ T cells in patients with PAH, consistent with previous reports (One of the limitations of sampling whole blood to derive transcripts is that samples comprise a mixed population of cells. We used established deconvolution methods to correct for potential confounding in RNA expression analysis. In support of the validity of this approach, we noted altered numbers of regulatory T cells and CD8 reports , 31, alt reports .This study does not assess the role of post-translational modifications in the pathogenesis of PAH, which could add further information to the circulating transcriptome. It is important that further mechanistic studies consider the role of the genes highlighted in this study in the tissues of primary interest, namely, the lung and heart. The study design included more patient samples than control subjects to account for the higher heterogeneity typically observed in patient populations. This heterogeneity is observed in the overlap in box plots of individual RNA levels or scores between subsets of patients with PAH and control subjects and emphasizes the importance of using any molecular markers in combination with best practice clinical assessments. We chose to use LASSO regression modeling not only as it is known to perform well in these kinds of data sets but also because it is widely applied and often easier to interpret than other methodologies. Another consideration is that the patients studied here were prevalent cases. Moreover, all the patients recruited had a clinical diagnosis of idiopathic, heritable, or drug-induced PAH. It would be of interest to sample patients with other presentations of PAH and other cardiopulmonary diseases to better understand the clinical utility of our RNA signature.In summary, we report a whole-blood RNA profile that distinguishes patients with PAH from healthy control subjects and reflects disease severity. Integration with genomic data suggests that SMAD5 is important in the development of PAH, prioritizing restoration of normal SMAD5 function as a target for therapeutic intervention.


The role of NF-κB in all diseases characterized by an inflammatory process, from cancer to autoimmune diseases, is known, but—precisely because it is involved in many diseases—this transcriptional factor continues to attract scientific research and the new knowledge that emerges is fundamental in highlighting the therapeutic potential that this factor can have in the various diseases in which it is involved.In this Special Issue, we have published reviews or experimental papers showing significant advances in the field of NF-κB’s role in many diseases.4 and resulting in an acute liver injury. The authors show that the polydeoxyribonucleotide (PDRN), an adenosine A2A receptor agonist, suppresses the secretion of pro-inflammatory cytokines and inhibits apoptosis through NF-κB inactivation. The authors conclude that PDRN could be a promising therapeutic agent for acute liver damage [Regarding research articles, we present an interesting contribution by Ko et al. about the role of NF-κB in the inflammatory process caused by CClr damage .With a particular focus on the role of NF-κB in the inflammatory process, Lyu et al. prove that, in canine macrophages, the inflammatory responses dependent on NF-κB can be regulated by heat shock protein 70 (Hsp70). Both proteins can therefore be considered potential therapeutic targets in animal species such as the dog .The first study to investigate whether the canonical, non-canonical, or atypical NF-κB activation pathways may be responsible for the higher activation of NF-κB observed in preeclamptic placentas has been published in this Special Issue. Preeclampsia (PE) is a multisystem disorder that affects 5–8% of all pregnancies worldwide, in some cases with a fatal outcome. The authors conducted the study on 268 cases (130 preeclamptic women and 138 controls) and indicate that, in the preeclamptic placentas, NF-κB activation occurs by mechanisms in which inhibitors of NF-κB such as IκBα or IκBβ may play a key role [D’Ignazio et al. highlight the role of the HIF-1β isoform in the NF-κB-mediated hypoxia response. HIF-1β, in fact, controls TRAF6 expression in human cells and also in Drosophila melanogaster. Thus, given that HIF-1β is required for full NF-κB activation in cells following canonical and non-canonical stimuli, the authors conclude that HIF-1β is an important regulator of NF-κB with consequences for homeostasis and human disease .Another important study on the role that many inflammatory mediators play in gamma irradiation-induced damage also focuses on the NF-κB pathway. The authors examine cell viability, reactive oxygen species (ROS), nitric oxide (NO) and glutathione (GSH) production, lipid peroxidation, DNA damage, inflammatory cytokine levels, and NF-κB pathway activation in human keratinocytes (HaCaT) and foreskin fibroblasts (BJ) after exposure to gamma radiation of 20 Gy. Contextually, they also examine the survival rate, levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the blood, and p65 and phospho-p65 expression in mice after exposure to gamma radiation. In both cases, treatment with celastrol, an NF-κB pathway blocker and antioxidant, blocks the inflammatory response by reducing oxidative DNA damage and lipid peroxidation in cells, as well as preventing excessive inflammatory responses in mice, in which celastrol increases the overall survival rate [Moreover, in the following article, the authors evaluate the role of NF-κB in inflammation, in particular in autoimmune disease, using relapsing–remitting experimental autoimmune encephalomyelitis (rEAE) in a mouse model that closely resembles relapsing–remitting multiple sclerosis in humans. Lunin et al. show that the reductions in the anti-inflammatory effects of thymulin (due to plasma cytokine level reduction both in the early and late stages of rEAE and decreases in NF-κB and SAPK/JNK cascade activation) are more marked when the thymic peptide thymulin is bound to polybutylcyanoacrylate (PBCA) nanoparticles. Lunin et al. show that the reduction in the anti-inflammatory effects of thymulin (both due to the reduction in the level of plasma cytokines in the early and late stages of rEAE and the decrease in cascade activation of NF-κB and SAPK/JNK) is more marked when the peptide thymus thymulin is bound to polybutyl cyanoacrylate (PBCA) nanoparticles.Thymulin and other thymic peptides, in fact, are characterized by their very low half-life in plasma (less than 10 min) and in vivo data show that orally administered poly-butylcyanoacrylate (PBCA) nanoparticles containing the thymic hormone thymopentin had similar effects to intravenous thymopentin, but with enhanced bioavailability due to the nanoparticles .The other four contributions are reviews. Ramadass et al. focus on small molecules, inhibitors of the NF-κB pathway, from cell surface receptor stimulation to nuclear signaling. The authors not only describe their clinical characteristics, but also their stage in clinical trials. This review is important because it helps to highlight the many possibilities of therapeutic choices that affect NF-κB, especially via small molecules with multiple modes of action .Betzler et al. explore recent evidence regarding the association of NF-κB and immune checkpoint expression and examine the therapeutic potential of inhibitors targeting either NF-κB directly or molecules involved in NF-κB regulation, in combination with the immune checkpoint blockade. In fact, there are still a significant number of tumor patients that do not benefit from an approach consisting of the immune checkpoint blockade alone. NF-κB inhibition can increase the immune checkpoint blockade.Leading to better patient responses, as well as different delivery systems like nanodrugs or hydrogels, the application of anti-PD-1mAb together with NF-κB inhibitors reveals promising results in relation to delivery efficacy and tolerability in preclinical models .The subsequent review examines different pharmacological approaches to reducing the expression/activation of NF-κB and thus restoring chemo-sensitivity in three tumor models characterized by innate or acquired  in particular triple-negative breast cancer (TNBC), hepatocellular carcinoma (HCC), and acute myeloid leukemia (AML), given NF-κB’s key role in inducing MDR. The authors also analyze the latest scientific evidence found by other groups, the most significant clinical trials regarding NF-κB, and new perspectives on the possibility of considering this transcriptional factor a valid drug target in neoplastic diseases .The last review focuses on the role of NF-κB-inducing kinase (NIK), the essential upstream kinase that regulates the activation of the noncanonical NF-κB pathway, in regulating immunity and inflammation, and presents recent studies investigating the therapeutic potential of NIK inhibitors in various diseases .This Special Issue is composed of contributions that extend their scope to other topics, while always keeping NF-κB as their central focus. For this reason, this Special Issue enriches our knowledge on the role of this transcription factor in various diseases. This special issue is made up of different contributions by topic, always keeping NF-κB in the center of attention. For this reason, this special issue enriches our knowledge on the role of this transcription factor in various diseases.


In vitro neutralizing antibodies have been often correlated with protection against Rift Valley fever virus (RVFV) infection. We have reported previously that a single inoculation of sucrose-purified modified vaccinia Ankara (MVA) encoding RVFV glycoproteins (rMVAGnGc) was sufficient to induce a protective immune response in mice after a lethal RVFV challenge. Protection was related to the presence of glycoprotein specific CD8+ cells, with a low-level detection of in vitro neutralizing antibodies. In this work we extended those observations aimed to explore the role of humoral responses after MVA vaccination and to study the contribution of each glycoprotein antigen to the protective efficacy. Thus, we tested the efficacy and immune responses in BALB/c mice of recombinant MVA viruses expressing either glycoprotein Gn (rMVAGn) or Gc (rMVAGc). In the absence of serum neutralizing antibodies, our data strongly suggest that protection of vaccinated mice upon the RVFV challenge can be achieved by the activation of cellular responses mainly directed against Gc epitopes. The involvement of cellular immunity was stressed by the fact that protection of mice was strain dependent. Furthermore, our data suggest that the rMVA based single dose vaccination elicits suboptimal humoral immune responses against Gn antigen since disease in mice was exacerbated upon virus challenge in the presence of rMVAGnGc or rMVAGn immune serum. Thus, Gc-specific cellular immunity could be an important component in the protection after the challenge observed in BALB/c mice, contributing to the elimination of infected cells reducing morbidity and mortality and counteracting the deleterious effect of a subneutralizing antibody immune response. Rift Valley fever virus (RVFV), a mosquito-borne bunyavirus, is widely distributed in Sub-Saharan countries, Egypt and the Arabian Peninsula, causing disease in both humans and livestock . RVFV isIn order to control RVF it is generally accepted that the induction of neutralizing antibodies is an important correlate of protection . TherefoIn this work we show that rMVA vaccines expressing independently versions of the glycoprotein Gc or Gn were able to confer substantial protection in mice, albeit inducing no detectable in vitro neutralizing antibody responses. Rather, the protection observed upon challenge was related to a strong T-cell response, which appeared more prominent against Gc epitopes. Intriguingly, the immune response elicited by the rMVA vaccine encoding both glycoproteins or glycoprotein Gn endowed the serum with the capacity to exacerbate disease when a serum:virus mixture was passively transferred to naive mice, as shown in experiments using either mouse or sheep immune sera obtained from rMVA vaccinated animals. It therefore appears that evoking a strong cellular response counteracts the failure in inducing protective humoral responses when an MVA vaccine is used. Conversely, the absence of an effective cell-mediated immune response may lead to disease exacerbation when subneutralizing antibody responses are elicited. These results warrant the optimization of our rMVA vaccines towards the induction of optimal humoral responses.d restricted CD8+ T cell epitope from Plasmodium berghei circumsporozoite protein (pb9) and an antiV5 monoclonal antibody recognition sequence. The plasmid for MVA construction also includes GFP as a reporter gene under the control of the vaccinia p11 late promoter. Both shuttle vectors were transfected into DF-1 cells (ATCC-CRL-12203) using lipofectamine 2000 , then infected with parental MVA and homologous recombination allowed the insertion of either Gn ectodomain (eGn) or Gc ORFs and the GFP marker gene at the TK locus of the MVA. Three consecutive rounds of green plaque purification were performed in order to obtain a pure preparation of each recombinant virus. The recombinant viruses (named rMVAGn and rMVAGc) were then further expanded in DF-1 cells. Semipurified, concentrated, virus preparations were obtained upon ultracentrifugation of infected cell extracts in a 36% sucrose cushion. The sucrose-purified virus fractions were titrated into DF-1 cells and stored at −80 °C until use.The rMVA-GnGc recombinant virus generated by homologous recombination of wild type MVA DNA and a plasmid construct encoding RVFV-MP12 glycoproteins (GB accession: DQ380208.1) into the TK locus of MVA was described previously . For theExpression of recombinant RVFV glycoproteins was analyzed by western blots of infected cell lysates using either specific antiGn or Gc antibodies  or a monCells were grown in either multi-well 96 (MW96) plates or in glass coverslips (CS) and infected with the recombinant MVA viruses at a multiplicity of infection (MOI) of 1. 24 h after infection the cells were fixed and permeabilized with 100% ice-cold methanol (MW96) or fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton-X100 (CS). Fixed cells were blocked with 10% FBS in PBS (10% blocking solution) for 30 min at room temperature (rt). AntiV5tag mAb, glycoprotein specific antibodies or antibodies specific to ER and Golgi proteins calreticulin and human mannosidase II (Bio-Rad’s AHP516 and AHP674 antibodies) were incubated for 1 h at rt in 2% blocking solution with 0.01 Tween-20. After three serial washing steps with PBST Alexa 488 conjugated antimouse, or Alexa-Fluor 594-conjugated antirabbit or antigoat mabs (Thermo) were incubated for 30 min at rt. Stained cells in MW96 were visualized using a Zeiss AX10 inverted fluorescence microscope . Stained CS preparations were mounted onto glass microscopy slides with or without DAPI staining (Thermo Fisher) and were visualized analyzed in a Zeiss LSM880 confocal laser microscope. Images were further processed using the Zen Zeiss software.For immunoprecipitation, monolayers of Vero cells were infected with RVFV-MP12 virus in the presence of 300 µCi/mL of [S35]-Methionine-Cysteine solution . After 24 h the cells were lysed in radioimmunoprecipitation assay buffer  and 20 µl of pooled sera from mice vaccinated with different rMVA viruses were incubated for 1 h at room temperature in a rotary shaker. Paramagnetic protein-G beads (Thermo Fisher) were added and incubated for an additional hour. The immunocomplexes were washed three times with the RIPA buffer and then separated by 12% SDS-PAGE. Fixed gels were subjected to fluorographic enhancement using Amplify solution (GE). After drying the gels were exposed to X-ray film.7 pfu of sucrose-cushion purified rMVA in phosphate-buffered saline (PBS). One or two weeks postvaccination, blood samples were taken either for neutralization assays (serum) or IFN-γ ELISpot (PBLs), and splenocytes for ex-vivo IFNγ ELISPOT at 7 or 14 dpi (n = 4). The remaining mice (n = 5), together with additional groups of unvaccinated BALB/c mice and immunized with nonrecombinant MVA , were all challenged intraperitoneally with 103 plaque-forming units (pfu) of the South African RVF virus strain 56/74 [50). Serum samples collected at later times postinfection in surviving mice were analyzed for the presence of neutralizing antibodies as described below. Vaccine efficacy estimation was evaluated in terms of morbidity and mortality monitoring daily over three weeks. All surviving mice were culled after 21 days of follow-up. Procedures involving animals received institutional approval (INIA’s ethics and biosafety Committee) as well as granted permits from regional veterinary authorities (Comunidad de Madrid PROEX 108/15).Groups of 5–10 BALB/c mice eight to ten weeks-old  were immunized intraperitoneally with 10in 56/74 . The immin 56/74 . BrieflySerum neutralizing antibody titers were measured in Vero cell monolayers by serial dilutions of serum mixed with an equal volume of medium containing MP-12 RVF virus strain and incubated for 1 h at 37 °C. After 72–96 h the cells were fixed and stained in a solution containing 10% formaldehyde and 2% crystal violet in PBS. The neutralization titer defined as the highest serum dilution at which cell lysis was reduced by 50% relative to cells incubated with RVF virus only. The assays were performed in triplicate and scored by an operator blinded to the vaccination regimen.Viral glycoprotein-specific T cells were measured by ex vivo IFNγ ELISPOT assay on splenocytes and pooled PBLs as described . Gn, Gc-3 pfu. Animals were monitored for clinical signs and mortality during three weeks and were weighed daily to quantify the extent of morbidity after challenge. Additionally, serum from sheep immunized with the rMVAGnGc vaccine, rMVA or from mock vaccinated was also pooled and passively transferred to mice.The sera for passive transfer protection studies were generated by pooling sera from mice immunized with the different rMVA constructs expressing the same antigens. Serum pools were prepared from day 14 post immunization and analyzed by virus neutralization and immunoprecipitation assays. As a positive control, antiRVFV immune mouse serum was used while antihuman adenovirus 5 (AdHu5) pooled mouse serum  was used as a negative control serum. For passive protection experiments each serum pool was ten-fold diluted in the virus inoculum used to challenge each group of 5 animals. 100 µL of each virus/serum mixture was injected intraperitoneally into adult female BALB/c mice. The virus challenge dose per mouse corresponded to 5 × 1010 to limit the range of variation found among individual mice. All analyses were done using the GraphPad 6.0 software . Differences were considered significant when p value <0.05The log rank (Mantel–Cox) test was used to check for differences in survival analysis following RVFV challenge. Individual ELISPOT values were determined by subtracting background values obtained after stimulation with media only. Statistical significance was calculated by one-way analysis of variance (ANOVA) transforming ELISPOT counts to logExpression was assessed by western blot analysis A. Both g2 = 09.503; df = 3; p = 0.023) when compared to the control MVA vaccine (Mantel–Cox log-rank test).The protective ability of a single dose of our rMVA vaccines was tested in BALB/c mice A. Mice ib-haplotype). The sensitivity of this mouse strain to the RVFV challenge is higher than that of the H-2d BALB/c strain (our unpublished data). Survival rates upon the RVFV challenge in the 129SvEv mice immunized with rMVAGnGc reached 80% with only one animal dying at 5 dpi. Contrarily, to what was observed in the BALB/c experiments, all of the 129SvEv mice that were vaccinated with rMVAGc or rMVAGn died upon challenge, with a slight delay in mortality in the rMVAGc group with respect to the MVA control group .Since the protective effect of our rMVAGnGc single dose vaccine relies mainly in the induction of specific CD8+T-cell responses  we questol group B. DifferWe had previously reported the low level of in vitro neutralizing antibody induction induced by a rMVAGnGc vaccine in BALB/c mice and sheep ,18. As e50 titer of 1:20 indicating that the observed neutralization at this dilution could be unspecific. When a more stringent neutralization determination was applied , no single prechallenge serum showed neutralization in all microtiter wells (not shown). Upon RVFV 56/74 challenge, all surviving animals showed elevated neutralization titers reaching around 3 logs. Although these data could indicate a successful priming of the vaccines, a similar titer observed in a surviving mouse from the control group ruled out this possibility. Interestingly, the mean postchallenge neutralization titer of the rMVAGnGc vaccinated 129EvSv mice was slightly higher, in agreement with the two mice showing prechallenge neutralization titers over the sensitivity limit. In spite of the lack of a clear in vitro neutralization activity, both vaccines induced antibodies able to label RVFV-MP12 infected cells as shown by indirect immunofluorescence . The rest of mice, either vaccinated with rMVAGn or rMVAGc did not show titers above the detection limit in any of the mouse models used. Moreover, one animal from the MVA control group showed a VNTrescence B. In therescence B. Howeverescence C.ELISPOT assay using 14 dpi pooled peripheral blood leukocytes (PBLs) from BALB/c mice immunized with rMVAGc showed the highest numbers of IFN-γ secreting cells upon restimulation with two different Gc-specific, MHC-I-restricted, peptides #13 (SYKPMIDQL) and #14 . The rest of the groups showed lower numbers of IFN-γ secreting cells, with the only exception of the rMVAGn group restimulated with the pb9 control peptide. The highest number of IFNγ secreting cells was also found in the MVAGc group in an ELISPOT assay using splenocytes collected at 7 dpi B or 14 d3 pfu/mouse) was preincubated in the presence of each serum pool  for 30 min, prior to the inoculation of mice. All mice that received the challenge dose in the presence of RVFV convalescent serum survived with no clinical display nor significant weigh loss  and eventually recovered from infection . Of note, the surviving mouse from the rMVAGnGc group had no conclusive viremia determination but it did not seroconverted (not shown), suggesting that this mouse was not efficiently infected.In order to gain insights in the role of the humoral response induced by the rMVA vaccines, a passive serum transfer experiment was designed. In this experiment individual sera from BALB/c mice collected 14 days after immunization with the different rMVA vaccines were pooled. The challenge virus dose  in the mice transferred with rMVAGnGc serum with respect to the serum from naive sheep . These data were suggestive of an exacerbating disease effect induced by the serum from animals vaccinated with rMVAGnGc or rMVAGn vaccines. Intriguingly, the extended survival in the rMVA control group was totally unexpected, indicating that the protective effect in the mice was not related to the presence of antiRVFV specific antibodies. In order to check whether the enhanced pathogenic effect of the rMVAGnGc serum was not exclusive of the mouse immune serum, sheep serum pooled from a previous rMVAGnGc vaccination experiment  was alsove sheep C. Taken We proposed previously that the protective ability of a recombinant MVA vaccine encoding GnGc antigens relied mostly in T-cell immune responses in the absence of a strong in vitro neutralizing antibody response . ApparenOne of the most striking findings in this work is the exacerbating effect of some rMVA immune serum in infectivity. This was somewhat unexpected but may help to explain our previous observations in experiments conducted to evaluate the efficacy of the rMVAGnGc vaccine in sheep . An indiIn conclusion we confirmed the possibility of protecting mice against a lethal RVFV challenge without induction of neutralizing antibody responses, stressing the importance of cell-mediated immune responses in protection. Most importantly, failing in inducing proper neutralizing antibody responses may result in enhanced pathogenesis when the cell mediated immune response is impaired or absent.


A growing proportion of head and neck cancers (HNC) result from HPV infection. Between HNC aetiological groups (HPV positive and HPV negative) clinical evidence demonstrates significantly better treatment response among HPV positive cancers. Cancer stem cells (CSCs) are identified in HNC tumour populations as agents of treatment resistance and a target for tumour control. This study examines dynamic responses in populations of a CSC phenotype in HNC cell lines following X-irradiation at therapeutic levels, and comparing between HPV statuses. Variations in CSC density between HPV groups showed no correlation with better clinical outcomes seen in the HPV positive status. CSC populations in HPV positive cell lines ranged from 1.9 to 4.8%, and 2.6 to 9.9% for HPV negative. Following 4 Gy X- irradiation however, HPV negative cell lines demonstrated more frequent and significantly greater escalation in CSC proportions, being 3-fold that of the HPV positive group at 72 hours post irradiation. CSC proportions of tumour populations are not fixed but subject to change in response to radiation at therapeutic dose levels. These findings imply a potential effect of aetiology on radio-responsiveness in CSCs, illustrating that clonogen treatment response may be more informative of therapy outcomes than inherent population density alone. Two principal aetiologies of HNSCC are those cancers associated with agents such as tobacco and alcohol and those resulting from the human papillomavirus (HPV) infection, which displays an anatomical preference for the base of tongue and tonsils, resulting in oropharyngeal squamous cell carcinoma (OPSCC)3. The most important distinction between these aetiologies is the significantly better treatment responses shown by HPV positive HNSCC, where the 5-year overall survival rate is around 80% compared to 50% for HPV negative HNSCC5. Moves toward more individualised treatment for HNSCC patients in terms of HPV status are hampered however, largely because the mechanisms and predictability of different treatment sensitivities according to HPV status remaining poorly understood8.Head and neck cancers are invasive carcinomas of the mucosal epithelium involving the complex anatomy of this area and frequently requiring radiotherapy as part of an approach aimed at tumour control whilst conserving normal tissue function. Globally, around 3.8% of cancers are head and neck cancers of which approximately 95% are squamous cell carcinomas (HNSCC)9. They demonstrate changes in divisional dynamics that can accelerate tumour re-population and are the numerical beneficiaries of tumour plasticity whereby de-differentiation of non-CSCs further increases the CSC population14. Consequently, therapy needs to account for CSC elimination to achieve tumour control16. CSCs have been identified in HNSCC tumour populations by several cellular markers tested by serial xenotransplantation of which CD44 and aldehyde dehydrogenase (ALDH) are found to be markers of stemness in both HPV statues21. Density of tumour CSC populations is recognised as a strong prognostic marker in a range of cancers22 but evidence of CSC density in HNSCC, by HPV status, is conflicting and fails to correlate with the better treatment outcomes of the HPV positive group25. These mixed results strongly imply additional factors are involved in CSC treatment response among HNSCC that result from HPV infection, and the prognostic interpretation of tumour CSC density may depend on HNSCC aetiology. Greater radiosensitivity and limited DNA repair following irradiation has been demonstrated in HPV positive tumours in pre-clinical studies28. Differences in the response to treatment according to HPV status in HNSCC has been widely researched, but data on the role played by CSCs, in terms of their HPV status, is scarce. What role CSCs play, as a result of HPV status, in response to treatment through radioresistance or the plasticity of tumour populations to replace them, remains unclear.Cancer stem cells (CSCs) are a small but critical subset within tumour cell populations, having unlimited capacity for self-renewal and generation of differentiated progeny. CSCs are mediators of metastases and recurrence following therapy and the most treatment resistant of tumour cellsin vitro study was on the quantitative evaluation of cell line dynamics during fractionated radiotherapy among HPV positive and negative aetiologies alike. Our previous work in this area, as a pilot study, examined only one cell line each HPV status, as such being insufficient to draw comparison between aetiologies, but suggestive of need for further investigation29. The current work comprises of data from 6 cell lines and provides valuable additional information, enabling not only qualitative but also quantitative comparison between HNSCC aetiologies by an examination of dynamic responses among CSCs to therapeutic levels of radiation in terms of their HPV status. While general understanding of HPV positive and negative responses are known, dynamic quantitative data for up to 6 cell lines has not been reported in literature so far30. This study therefore progresses current knowledge from generic toward specific where findings demonstrate significant differences in CSC population response to X-ray irradiation between the HPV groups, implying greater repopulating ability in HPV negative HNSCC.The aim of this study has been to investigate behavioural responses of CSCs to irradiation, in terms of their HPV status, measuring inherent CSC proportions of HNSCC cell lines and changes in CSC populations in response to irradiation as a function of HPV status. The key focus of this in vitro experimental study measured a) the inherent proportions of the CSC phenotype CD44+/ALDH + in HNSCC cell lines, b) changes in CSC proportions of population over time following 4 Gy X-ray dose, c) parallel assays to compare cell population response in T25 and 6 well plates .This 31. Cells were cultured in T25 flasks using Dulbecco’s Modified Eagle’s Medium with 4500 mg/L glucose,  supplemented with with 10 mM HEPES, 10% foetal bovine serum (FBS), 100 U/mL penicillin and with 0.1 mg/mL streptomycin . Cells were incubated in humidified atmosphere of 5% CO2 at 37 °C. Cell lines were tested for mycoplasma used MycoAlert detection kit  and were negative.Six HNSCC cell lines were selected, 3 of each HPV status. UM-SCC-1, UM-SCC-17a, UM-SCC-22a and UM-SCC-47 were purchased through Merck Millipore . UPCI-SCC-090 (ATCC CRL-3239TM) and UPCI-SCC-154 (ATCC CRL-3241TM), both HPV positive, were purchased from ATCC (Manassas VA). UM-SCC-1, UM-SCC-17a and UM-SCC-22a are HPV negative and UM-SCC-47, UPCI-SCC-090 and UPCI-SCC-154 are HPV positive  to achieve full scatter conditions. Flasks were filled with media (21 mm depth over the cell monolayer) to achieve electronic equilibrium at the cell layer. One T25 flask of each cell line, for each analysis time point  was irradiated with a 4 Gy dose. 4 Gy was chosen as the treatment dose as it is low enough to be at the upper end of what a single fractionated dose might be in therapy, and sufficiently high to produce cell killing and a clear response among survivors. Matching controls for each flask were sham irradiated. Media was changed post irradiation and flasks for assay were incubated with cells remaining in situ until analysis.Cell cultures were irradiated in T25 flasks using a 160 kV, 25 mA, RS2000 X-ray cabinet irradiator  calibrated using the AAPM TG61 protocolCSCs were putatively identified in the 6 HNSCC cell lines by 2 cellular markers: cluster of differentiation 44 (CD44), a cell surface protein and adhesion molecule, and aldehyde dehydrogenase (ALDH), a metabolic enzyme. CD44 expression was determined by anti-mouse/human CD44 antibody, clone IM7, fluorophore APC . ALDH expression was measured using Aldefluor . Gating for CD44 was based on the APC isotype control and diethylaminobenzaldehyde (DEAB) was used for gating of ALDH expression Fig. . Cells wBaseline proportions of CSC populations were established through concurrent flow cytometry of the 6 unirradiated cell lines for their CD44+/ALDH + population. Measurements of CSC proportions in unirradiated and irradiated cell line populations were conducted 7 times at 24, 48 and 72 hours following a 4 Gy X-ray dose. Time points at 1, 2 and 3 days were chosen as they reflect normal intervals between radiotherapy fractions during which CSC density may increase or potentially re-establish populations.in situ following irradiation until analysis, parallel assays were conducted to test for significance of any difference in results. For comparison, cells in T75 flasks, also under 21 mm of media and associated sham irradiated controls, were irradiated with 4 Gy then detached using TrypLE Select  centrifuged and re-plated in 6 well plates until concurrent analysis with cells from T25 flasks.To test for associative effect on CSC proportions as a result of cells being contained Flow cytometry data was analysed in Microsoft Excel and Prism v7.01 (GraphPad Software Inc. La Jolla CA). Data from repeated cell line analysis (n = 7) was reported as mean and SEM for each cell line. Multiple t-tests were used to determine significance in CSC population change following 4 Gy dose. Two way ANOVA and Sidak’s multiple comparisons were used to test significance for difference in ratio of response. Unpaired t-tests were used to compare CSC data for control cells of T25 versus 6 well plate methods and irradiated cells. Significance considered to be p < 0.05 .Proportions of putative CSCs in untreated cultures of 6 HNSCC lines were determined by staining for CD44 and ALDH. The proportions of CSC varied between 2% to 10% of the total cell population. Large variations in CSC density was observed among the cell lines, irrespective of HPV status  at 72 hours following irradiation  and another CSC, representing self-renewal. But in response to tumour volume loss from irradiation, cellular signalling may switch the CSC divisional process to a symmetrical one where both daughter cells are CSCs, rapidly increasing their proportion in the tumour population and thus their potential for proliferation37. Furthermore, plasticity among progenitor and differentiated cells, in response to irradiation, can increase CSC populations by dedifferentiation where these cells acquire the CSC phenotype and the capacity for self-renewal13. Observed increases in CSC proportions demonstrate that CSC numbers are not static but may be responsive to therapeutic levels of irradiation where a significantly greater effect was observed in the HPV negative group. This observed difference suggests a potential difference in radio-responsiveness among HNSCCs depending on aetiology, and warrants further investigation. The fact that recurrent primary HNSCC tumours are the most common cause of mortality points to the importance of our finding that HPV status seems to have differential effects on the relative proportions of putative CSCs among the entire cell population of HNSCCs after a single 4 Gy dose of X-irradiation and thus may also affect the relative repopulating ability of CSCs according to their HPV status38.To test for differences in the radio-responsiveness of the CD44+/ALDH + phenotype of HPV positive versus negative HNSCC cells, cell cultures were irradiated with a 4 Gy dose and analysed for changes in the proportion of this phenotype over intervals of 24, 48 and 72 hours, reflecting time intervals between fractionated doses in external radiotherapy. Resulting data is novel in that it provides a quantitative comparison of dynamic population responses among HSCC CSCs, by their HPV status, at therapeutically relevant dose and time points following irradiation. Among irradiated cell cultures, only 1 of the 6 cell lines (UM-SCC-22a) showed any increase in CSC density at 24 hours. Although CSC density increased among the HPV positive lines, UPCI-SCC-154 and UPCI-SCC-090, the HPV negative populations of HNSCC cells more often had significantly greater increases in the proportion of CSCs during the 3 days after irradiation. To compare how differences in HPV status might affect the overall extent to which CSC density responded at 24, 38, and 72 hours after irradiation, we calculated the ratio of the proportions of putative CSCs post-treatment to baseline (untreated) cell line populations, at each time point for either the HPV positive or the HPV negative HNSCC cell lines. We found that this ratio increased steadily during the 3 days after irradiation so that the proportional change in CSC density of the irradiated HPV negative HNSCC cells was significantly greater than that of the irradiated HPV positive cells by day 3. The increased proportions of CSCs over the 3 days may indicate that several different biological processes are at work including preferential survival, altered divisional dynamics and/or cellular plasticity by de-differentiation. The danger of clonogen-driven acceleration in tumour repopulation, in response to sublethal or delayed doses of radiation, has been earlier described by Withers, in situ in T25 flasks demonstrated less range of error than the 6 well plate method and was felt to introduce fewer confounding effect from cell handling.Comparison of protocols (T25 flask vs 6 well plate) for potential effects on CSC counts, found one significant difference in results from these methods among the 6 comparisons of means. This may be indicative of the extent to which methods can influence this data and needs to be considered in the interpretation of results. The measurement of CSCs from cells remaining inherent CSC densities among the investigated HNSCC cell line populations did not associate with HPV status;the study compared changes in CSC densities within the cell line population, by HPV status, in response to radiation;quantitative differences in CSC populations were shown using 6 cells lines, even amongst HPVpositive and negative sub-groups, based on their origin;the behavioural response of putative CSCs among the 6 cell lines investigated demonstrated elevations in CSC densities following a 4 Gy dose that were significantly greater in the HPV negative group, potentially indicating stronger repopulating ability;in vitro, in vivo and in silico (modelling studies) that require specific quantitative inputs.the dynamic quantitative data obtained for the 6 cell lines facillitates progress of knowledge from generic to specific, as only qualitative studies have been reported so far in the literature; this information is important for future There is very little quantitative data in the scientific literature on the cellular dynamics of cancer stem cells originating from HNSCC, in response to therapeutic levels of X-irradiation, which expressly compares CSCs by HPV status. This work provides additional insight using data from 6 cell lines (3 HPV positive and 3 HPV negative), thus enabling for the first time quantitative comparison between HNSCC aetiologies by analysis of dynamic responses among CSCs to therapeutic levels of radiation in terms of their HPV status. The main highlights of the current work are as follows:Data on responsiveness of HNSCC clonogens to therapeutic levels of radiation, comparing HPV statuses, is of vital interest in finding better clinical applications. Given the number of clinical trials investigating dose de-escalation for HPV positive HNSCC, quantification of intrinsic capacity for response to treatments by CSCs in both HPV statuses is of critical concern and value to informing approaches toward de-escalation.The differential response of CSC densities to therapeutic levels of X-ray dose, according to HPV status, warrants further studies. Apoptotic studies and FACS analysis, to measure preferential survival against proliferative of differential changes in phenotype proportions are required to measure their influence in resulting phenotype proportions over time following irradiation. Testing of intrinsic radiosensitivity by proliferative capacity following fractionated irradiation, using clonogenic assays of cell line populations, needs to be part of future work determine dynamic response to repeated dose. Any changes in cell survival, as a function of dose and HPV status, following repeated 4 Gy dose would provide data on changes to intrinsic radiosensitivity reflective of the therapeutic use of radiation.


GH1, ACE, ASIC3, HSPH1, MVD, BCL2, HIGD2A, CBFA2T3) that may be involved in physiological adaptations required to cope with environmental stressors that are typical of the North African area such as infectious diseases, extended drought periods, scarce food supply, oxygen scarcity in the mountainous areas and high-intensity solar radiation. Our data also point to candidate genes involved in transcriptional regulation suggesting that regulatory elements had also a prominent role in North African cattle response to environmental constraints. Our study yields novel insights into the unique adaptive capacity in these endangered populations emphasizing the need for the use of whole genome sequence data to gain a better understanding of the underlying molecular mechanisms.Natural-driven selection is supposed to have left detectable signatures on the genome of North African cattle which are often characterized by the fixation of genetic variants associated with traits under selection pressure and/or an outstanding genetic differentiation with other populations at particular loci. Here, we investigate the population genetic structure and we provide a first outline of potential selection signatures in North African cattle using single nucleotide polymorphism genotyping data. After comparing our data to African, European and indicine cattle populations, we identified 36 genomic regions using three extended haplotype homozygosity statistics and 92 outlier markers based on Bayescan test. The 13 outlier windows detected by at least two approaches, harboured genes (e.g.  The geographic proximity of North Africa to Europe makes it a likely contact zone between the two continents. Several genetic studies reported an old presence of African cattle ancestry in the genomes of Iberian cattle3 and a European ancestry in local Maghreb cattle6. Nomad pastoralism and tribal migrations prevented the division of North African cattle populations into clearly defined breed groups. Present-day indigenous cattle in Morocco, Algeria Tunisia and Libya belong to the Brown Atlas cattle. These are small-sized, sturdy, fairly compact animals with fine limbs, a short head and a straight to slightly concave profile. In these countries, Brown Atlas cattle populations, predominantly pasture-fed, are raised in a Mediterranean climate characterized by a winter rainfall and a hot dry summer during which live weight losses in adult cows can reach 20%7. In Egypt, indigenous cattle are medium sized, long-bodied animals, lean of musculature and lightly boned. They are raised either in desert or semi-desert regions characterized by a very arid Mediterranean climate and negligible rainfall. A number of ecotypes are recognised based on their geographical distribution. For instance, in Lower Egypt there are two local cattle populations, the Damietta is typically found in coastal sites and the Baladi or Baheri is widespread inland in the delta8. Overall, North African indigenous cattle are resistant to many of the diseases and parasites to which imported European cattle are susceptible7 resulting from a local environment-driven selection that occurred over hundreds of years. Adaptation to local conditions is expected to leave distinct signatures in the genome known as a “selective sweeps” owing to a rapid increase in the frequency of the desirable alleles or in the frequency of neutral markers in linkage disequilibrium with the favorable alleles9. Studies on signatures of selection focusing exclusively on North African cattle have never been reported before.Taurine cattle were first introduced to Africa through Egypt from the Fertile Crescent ~ 6500 years BP14. All these studies have used comparison of genomic patterns of SNPs variability between local and exotic breeds to identify genomic regions and genes that have undergone selective sweeps.The emergence of high-throughput single nucleotide polymorphism (SNP) genotyping and whole genome sequencing facilities coupled with the development of new genomic methodologies have enabled the screening of a large part of the genome to detect signatures of selection in livestock and domestic populationsiHS, Rsb and XP-EHH) and are based on the decay of haplotype homozygosity as a function of recombination distance. The fourth approach is a Bayesian method based on the differentiation of allele frequencies among populations.The main goal of this study was to investigate population structure and candidate positive selection signatures in North African cattle using genotype data from the Illumina BovineSNP50 BeadChip with comparisons against four European breeds, three African and two indicine populations. We applied four genome scan approaches to identify genomic regions putatively under selection: the first three methods are extended haplotype homozygosity (EHH)-derived statistics  to contextualize the genetic variation of North African cattle populations Fig. . The firBreed assignment to clusters using ADMIXTURE provided further insight into the genetic structure of North African populations. Figure FST values are found between CHE and GUE (FST ~ 0), CHE and TUNIND (FST = 0.002) and between GUE and TUNIND (FST = 0.003). Likewise, low genetic differentiation is observed between TID on one hand, GUE, CHE and TUNIND, on the other hand  while a higher FST is observed between these three breeds and BAL .Details of the level of pairwise genetic differentiation are reported in Supplementary Table We used the TreeMix software to model both population splits and gene flow between the 17 cattle populations. When no migration events were fit  and CHF  Figs. , 2, 3. WRsb and Cross-population Extended Haplotype Homozygosity (XP-EHH) statistics were computed at each SNP for each of the three comparisons (African (AFT)/North African, European (EUT)/North African, indicine (IND)/North African). Haplotypes estimated in each population were pooled, for each autosome, according to their group of origin. In total, 108, 334 and 86 haplotypes were considered as representative of African, European, and indicine ancestries, respectively.Rsb detected 427, 369 and 167 SNPs putatively under selection for AFT/North AFT, EUT/North AFT and IND/North AFT comparisons, respectively  distributed on BTA 03  and 19  . Overall, the 11 candidate genomic regions identified by at least two EHH-based methods, overlap with Quantitative Trait Loci (QTL) associated with traits for milk and meat composition, fertility and sexual precociousness, disease susceptibility (tuberculosis and respiratory diseases), stature and growth . Similarly, 109, 143 and 65 genes were jointly identified by Rsb and XP-EHH for each of the AFT/North African, EUT/North African, IND/North African comparisons, respectively of which 74, 97 and 50, respectively, could be mapped by DAVID Bioinformatics resources (https://david.ncifcrf.gov/). Gene Ontology (GO) analysis showed that AIG1  and P-loop containing nucleoside triphosphate hydrolase  InterPro protein functional groups were the two significantly enriched functional classes identified in the AFT/North AFT comparison  and G-protein coupled receptor signaling pathway  were the most enriched biological process (BP) terms identified in the EUT/North AFT comparison. Olfactory receptor activity  and serine-type endopeptidase activity  were the most enriched terms under molecular function (MF) in the same comparison . The third subgroup, represented by CHF and BIS, includes European-derived breeds. The phylogenetic network inferred by TreeMix corroborate these findings in that CHF and especially BIS are in clade with the European breeds while CHE, TID, TUNIND and GUE share the same branch and are much closer to African populations.Our results corroborate previous reportsiHS, Rsb and XP-EHH) and 92 outlier SNPs using Bayescan. Consistently with previous observations19, we observe little overlap between results obtained from each of the two types of approaches. Given that Bayescan assumes that the gene frequencies under any neutrally structured population model can be approximated by a multinomial Dirichlet distribution20 which would not be appropriate in a hierarchical population structure21 (as is the case for our North African sample), the 92 identified SNPs should be considered cautiously. Instead, we believe that the three EHH-based methods, which inter alia, can detect a wider range of selection scenarios22, are more suitable to our study design. These statistics take advantage of the reduction in haplotype diversity in the neighbourhood of a beneficial mutation due to a “hitch-hiking” effect. They measure the extended haplotype homozygosity which is defined as the probability of identity by descent for two randomly chosen haplotypes carrying a core haplotype of interest in an interval around a given locus, given that they have the same allele at the locus23. Unlike Rsb and XP-EHH, the iHS test has low power in identifying fixed sweeps because it requires the ancestral allele to be still segregating in the population24. Here, we identified a higher number of outlier windows using Rsb and XP-EHH compared to the iHS approach which might suggest, at first glance, that most of the candidate regions identified here have undergone a positive selection resulting in the (near) fixation of the favoured alleles across the populations. However, we believe that the low number of candidate regions identified by the iHS test is actually due to the fact that this approach searches for loci where a given high-frequency haplotype is much longer relative to all other haplotypes, yet in a soft sweep several long haplotypes will be present at the adaptive locus and thus not one haplotype will typically be much longer than all others25. Our hypothesis assumes that the majority of sweeps detected here are soft which is likely to be the case. Soft sweeps were shown to be widespread and account for the vast majority of recent environmental adaptation in several species such as Humans24. A common constraint of selection signature detection methods is the detection of false positives. One efficient way to reduce their number is to retain as outliers, those genomic regions detected by distinct methods26. Among the 36 genomic regions identified by EHH-based methods, 10 were detected by two tests and one candidate region was identified by all three tests. In addition, two other regions  identified by the Rsb EUT/North AFT comparison included two outlier SNPs detected by Bayescan. We particularly focused on genes located within these 13 genomic regions. In agreement with previous findings28, we observed that the three candidate regions jointly identified by the Rsb and XP-EHH tests in the EUT/North African comparison were significantly enriched for genes involved in olfactory receptor activity (21 genes) which might reflect the fact that selection has been relaxed around these genes in European breeds which are often raised in abundant food supply conditions. Two genes (OR2W3 and OR2L13) coincided with CNVs previously reported in cattle  belong to the S1A family of peptidases, a superfamily of proteolytic enzymes with a wide variety of biological functions in parasite infection30. Similarly, another relevant selection signature on the BTA19 jointly detected by iHS, Rsb and XP-EHH EUT/North African harboured several genes which are involved in immune response: CD79B, MILR1, PECAM1, MAP3K3 and TCAM1. The last two genes mediate NF-kappa-B activity which show evidence of positive selection in the African N’Dama cattle to alter in functions to effectively regulate the infection of cattle trypanosome31. Consistently, we also observed that outlier windows from AFT/North African and IND/North African comparisons included many genes associated with immune response and host defence such as TNFRSF11A, IRF8, MYO1G and several GTPases of immunity-associated protein (GIMAP) genes . Several of these genes  coincided with CNVs reported in cattle , blood vessel development and morphogenesis . These genes are expected to be involved in adaptation to extreme temperatures prevailing in several Northern African areas and/or to chronic hypoxia in the Atlas mountain ranges where the altitude varies between 900 and 4000 m7. Our hypothesis is consistent with the presence of three hypoxia-related genes  and three other genes involved in response to heat  in the relevant candidate regions , positive regulation of lactation (GO:1903489) and triglyceride biosynthetic process (GO:0010867) and was previously reported as being a candidate gene for dairy production traits in Braunvieh cattle15. Importantly, it has been suggested that elevated GH1 gene expression may constitute an adaptive response to the effects of scarce food supply in a sample of 163 human individuals from Benin35. We therefore suggest that this gene is particularly under positive selection across North African cattle populations as a consequence of important seasonal fluctuations in food availability characterizing the whole region.Many of our candidate regions harboured genes implicated in the adaptive immune response against microbial pathogens. For instance, the clearest sweep signal in the EUT/North AFT comparison detected on BTA07 (between positions: 41.06 and 43.62 Mb) with 13 SNPs (out of 32) exceeding the significance threshold, harboured 58 known genes amongst which six , harboured RNF152 gene which positively regulates Toll-like receptors (TLRs) which are important pattern recognition receptors that are critical for the defence against invading pathogens36. RNF152 gene was reported to be involved in local adaptations in the Ainu, a hunter-gatherer population of northern Japan37. Another relevant candidate region on BTA21  harboured four protein coding genes: SLCO3A1, SV2B, AKAP13 and KLHL25. The latter two genes were shown to be under positive selection in Creole cattle breeds38 while SLCO3A1 is associated with marbling score in the Montana Tropical Composite beef cattle39 and mediates inflammatory processes in intestinal epithelial cells through NF-kappa-B transcription activation in humans40. SV2B gene is among major genes enriched for the extracellular matrix (ECM) around the hair follicle in Changthangi goats41. ECM is considered important for regulating the structure, metabolism and signaling of dermal papilla cells which play key roles in hair follicle morphogenesis and regeneration42. Another candidate region on the BTA22  identified by the XP-EHH IND versus North AFT test harboured four genes  and among these, GADL1 gene is one of the genes involved in adaptive evolution of Anolis carolinensis introduced into the Ogasawara archipelago43. Gadl1−/− mice exhibited decreased anxiety, increased levels of oxidative stress markers, alterations in energy and lipid metabolism, and age-related changes44. STT3B is a catalytic subunit of hetero oligomeric oligosaccharyltransferase (OST), which is important for asparagine linked glycosylation. In mammals and plants, OSTs exhibit distinct levels of enzymatic efficiency or different responses to stressors45. OSBPL10 gene confers African-ancestry protection against dengue haemorrhagic fever in admixed Cubans46.Six out of the 13 relevant candidate regions identified in this study, harboured fewer than 15 known protein coding genes  as well as many non-coding RNAs including 9 small nucleolar RNAs (snoRNAs), 12 microRNAs (miRNAs), 10 small nuclear RNAs (snRNA) and 13 long noncoding RNAs (lncRNAs). In addition, many of the aforementioned genes  co-localize with known cattle CNVs. It is worth noting that CNVs encompassing a gene encoding a transcription factor has a greater phenotypic impact because it can affect both the coding sequence of the gene itself as well as the expression of downstream targets of that gene. From a selective standpoint, these findings suggest that natural selection has shaped North African cattle genome not only through variation in coding sequence but also through extensive regulation of gene expression occurring both at the transcriptional and post-transcriptional level. Lending further support to this hypothesis, the relevant candidate region on BTA24  harbours a single gene, CELF4, coding for an RNA-binding protein mainly expressed in central nervous system that regulates the expression of many genes co-transcriptionally or post-transcriptionally via interactions with mRNA47. Celf4-deficient mice have additional neurological abnormalities including hyperactivity and hyperphagia-associated obesity48. Similarly, the most relevant selection signal in the AFT/North AFT comparison  harboured one protein coding gene (PCDH7) which coincides with a known CNV  obtained from Flori et al.16 and Gautier et al.53. We also included genotyping data belonging to 9 other populations, representatives of European taurines (EUT) , African taurines (AFT)  and indicine (two populations: GIR and NEL) from Matukumalli et al.54. All genotypes were recovered from the web-interfaced genetic Diversity Exploration (WIDDE) database55. We performed a relatedness test between individuals within each population using PLINK56. The software calculates a variable called PI-HAT reflecting extended haplotypes shared between distantly related individuals. For European, indicine and African breeds, we removed closely related individuals if the PI-HAT value was greater than 0.25 which is a value roughly corresponding to relationships closer than grandsire-granddaughter. For the North African populations, in which natural service is commonly used rather than artificial insemination and are thus generally less inbred, we used a more stringent threshold and excluded one individual from each pair of individuals with a PI-HAT value > 0.1. In total, after relatedness filtering, 468 individuals including 204 North African animals, were available for the different analyses (Supplementary Table 2: 0.5). In total, 38,464 SNPs spread over all autosomal chromosomes were finally considered for population structure analyses.We combined Illumina BovineSNP50 BeadChip genotypes of 57 Brune de l’Atlas individuals (TUNIND) sampled from our previously published data57. Unsupervised hierarchical clustering was carried out for all populations using ADMIXTURE 1.23 software58. We ran ADMIXTURE with cross-validation for values of K from 2 through 17 (the number of populations) to identify the best value of K clusters. DISTRUCT software59 was then used to graphically display ancestry within each individual. The pairwise fixation index (FST) between populations was estimated using Genepop 4.6 software60. The patterns of population splits and mixtures were inferred using TreeMix61. First, we built a maximum likelihood tree of the 17 populations of the study with no migration events allowed and setting GIR as outgroup. Then, we built a phylogenetic tree of these populations and started adding migration events (modeled as edges) sequentially to the phylogenetic model. The migration edges were added until 99.93% of the variance in ancestry between populations was explained by the model. The residuals from the fit of the model to the data were visualized using the R script implemented in TreeMix.Population structure was inferred by PCA for African, European, indicine and North African populations using the adegenet R packageFST)62 and three extended haplotype homozygosity (EHH)-based tests  to detect signatures of selection within North African cattle. Bayescan, Rsb and XP-EHH analyses were performed for each of the three pairwise comparisons: North African cattle versus AFT, North African cattle versus EUT and North African cattle versus IND. Bayescan uses a reversible-jump Markov Chain Monte Carlo to separate locus-specific effects of selection from population-specific effects of demography. Outliers are those loci that require the locus-specific component to explain observed genetic diversity. For the Markov chain Monte Carlo (MCMC) algorithm we used 20 pilot runs of 5,000 iterations, a burn-in of 50,000 iterations, a thinning interval of 10  with a resulting total number of 100,000 iterations. To control the number of false positives, significant SNPs were defined by applying a q-value threshold of 0.05.To perform selection signature detection, we selected the individuals that are most representative of the ancestral North African cattle. This was done based on the results of model-based clustering results. We used the population differentiation based analysis implemented in BayeScan -0.5|]. As a prerequisite to the Rsb and XP-EHH computation, haplotypes were reconstructed from the genotyped SNPs using fastPHASE 1.467. The following options were used for each chromosome: -T10 -Ku60 -Kl10 -Ki10. Considering that Rsb and XP-EHH values are normally distributed, a Z-test was applied to identify significant SNPs under selection. Two-sided p value s were derived as pRsb =  − log10[1–2|Φ(Rsb)-0.5|] and pXP-EHH =  − log10[1–2|Φ(XP-EHH)-0.5|] where Φ (x) represents the Gaussian cumulative distribution function. In EHH-based tests, the maximum allowed gap between two SNPs was set to 500 Kb. We used 1-Mb sliding windows that partially overlapped 10 kb with adjacent windows to perform selection signature detection. A window is classified as putatively under selection when it contains at least 3, 4 and 4 markers exceeding the significance threshold of − log10  = 3 for iHS, Rsb and XP-EHH tests, respectively. Finally, we checked the overlap of the candidate genomic regions detected with at least two EHH-based approaches with the previously identified bovine Quantitative Trait Loci (QTL) available in the cattle QTL database . The overlaps were checked using QTL coordinates according to the Bos taurus genome assembly ARS-UCD1.2.Haplotype extended patterns were investigated using three metrics implemented in iHS, Rsb, XP-EHH) were interrogated for genes annotated to the Bos taurus genome assembly ARS-UCD1.2 using BioMart tool of Ensembl (https://www.ensembl.org/biomart/martview/c8fe3a69961a4088a55b7a249db7e2fa). Cattle structural variants which overlapped the genomic coordinates (in bp) of these relevant candidate selective sweep regions were retrieved using the same database. We have only considered structural variants of less than 8 Mb which corresponds to the maximum size that can be identified, from whole genome sequence data, by the pindel software . We used the online tool, Database for Annotation, Visualization and Integrated Discovery (DAVID) software version 6.8 (https://david.ncifcrf.gov/) for functional enrichment analysis of the genes retrieved from BioMart. GO enrichment analysis included two aspects: Biological Process and Molecular Function. For the GO functional groups and InterPro functional terms returned from DAVID functional analysis, we considered an adjusted Benjamini-corrected p value threshold of ≤ 0.05.Candidate genome region intervals detected by at least two EHH-based methods (Supplementary Information 1.