ISOTOPE: ISOform-guided prediction of epiTOPEs in cancer.

Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available at https://github.com/comprna/ISOTOPE.

A QTL for conformation of back and croup influences lateral gait quality in Icelandic horses.

BACKGROUND: The back plays a vital role in horse locomotion, where the spine functions as a spring during the stride cycle. A complex interaction between the spine and the muscles of the back contribute to locomotion soundness, gait ability, and performance of riding and racehorses. Conformation is commonly used to select horses for breeding and performance in multiple horse breeds, where the back and croup conformation plays a significant role. The conformation of back and croup plays an important role on riding ability in Icelandic horses. However, the genes behind this trait are still unknown. Therefore, the aim of this study was to identify genomic regions associated with conformation of back and croup in Icelandic horses and to investigate their effects on riding ability. One hundred seventy-seven assessed Icelandic horses were included in the study. A genome-wide association analysis was performed using the 670 K+ Axiom Equine Genotyping Array, and the effects of different haplotypes in the top associated region were estimated for riding ability and additional conformation traits assessed during breeding field tests. RESULTS: A suggestive quantitative trait loci (QTL) for the score of back and croup was detected on Equus caballus (ECA) 22 (p-value = 2.67 × 10- 7). Haplotype analysis revealed two opposite haplotypes, which resulted in higher and lower scores of the back and croup, respectively (p-value < 0.001). Horses with the favorable haplotype were more inclined to have a well-balanced backline with an uphill conformation and had, on average, higher scores for the lateral gaits tölt (p-value = 0.02) and pace (p-value = 0.004). This genomic region harbors three genes: C20orf85, ANKRD60 and LOC100056167. ANKRD60 is associated with body height in humans. C20orf85 and ANKRD60 are potentially linked to adolescent idiopathic scoliosis in humans. CONCLUSIONS: Our results show that the detected QTL for conformation of back and croup is of importance for quality of lateral gaits in Icelandic horses. These findings could result in a genetic test to aid in the selection of breeding horses, thus they are of major interest for horse breeders. The results may also offer a gateway to comparative functional genomics by potentially linking both motor laterality and back inclination in horses with scoliosis in humans.

Genome-wide association study for insect bite hypersensitivity susceptibility in horses revealed novel associated loci on chromosome 1.

Equine insect bite hypersensitivity (IBH) is a pruritic skin allergy caused primarily by biting midges, Culicoides spp. IBH susceptibility has polygenic inheritance and occurs at high frequencies in several horse breeds worldwide, causing increased costs and reduced welfare of affected horses. The aim of this study was to identify and validate single nucleotide polymorphisms (SNPs) associated with equine IBH susceptibility. After quality control, 33,523 SNPs were included in a Bayesian genome-wide association study on 177 affected and 178 unaffected Icelandic horses. We report associated regions in E. caballus (ECA) 1, 3, 15 and 18, overlapping with known IBH QTLs in horses, and novel regions containing several genes, together explaining 11.46% of the total genetic variance. For validation, three SNPs on ECA 1 and ECA X (explaining the largest percentage of genetic variance) within 1-mb genomic windows for IBH were genotyped in an independent population of 280 Exmoor ponies. The associated genomic region (152-153 mb) on ECA 1 was confirmed in Exmoor ponies and contains the AQR gene involved in splicing processes and a long non-coding RNA. This study confirms the polygenic nature of IBH susceptibility and suggests a role of transcriptional regulatory mechanisms (e.g., alternative splicing) for IBH predisposition in these horse breeds.

Genetics of Skin Disease in Horses.

Equine skin diseases are common, causing increased costs and reduced welfare of affected horses.Genetic testing, if available, can complement early detection, disease diagnosis, and clinical treatment and offers horse breeders the possibility to rule out carrier status. The mechanisms of complex disease can be investigated by using the latest state-of-the-art genomic technologies. Genome-based strategies may also serve as an efficient and cost-effective strategy for the management of the disease severity levels, with particular interest in complex traits such as insect bite hypersensitivity, chronic progressive lymphedema, and melanoma.

Exploring the genetics of trotting racing ability in horses using a unique Nordic horse model.

BACKGROUND: Horses have been strongly selected for speed, strength, and endurance-exercise traits since the onset of domestication. As a result, highly specialized horse breeds have developed with many modern horse breeds often representing closed populations with high phenotypic and genetic uniformity. However, a great deal of variation still exists between breeds, making the horse particularly well suited for genetic studies of athleticism. To identify genomic regions associated with athleticism as it pertains to trotting racing ability in the horse, the current study applies a pooled sequence analysis approach using a unique Nordic horse model. RESULTS: Pooled sequence data from three Nordic horse populations were used for FST analysis. After strict filtering, FST analysis yielded 580 differentiated regions for trotting racing ability. Candidate regions on equine chromosomes 7 and 11 contained the largest number of SNPs (n = 214 and 147, respectively). GO analyses identified multiple genes related to intelligence, energy metabolism, and skeletal development as potential candidate genes. However, only one candidate region for trotting racing ability overlapped a known racing ability QTL. CONCLUSIONS: Not unexpected for genomic investigations of complex traits, the current study identified hundreds of candidate regions contributing to trotting racing ability in the horse. Likely resulting from the cumulative effects of many variants across the genome, racing ability continues to demonstrate its polygenic nature with candidate regions implicating genes influencing both musculature and neurological development.

Inter- and intra-breed genome-wide copy number diversity in a large cohort of European equine breeds.

BACKGROUND: Copy Number Variation (CNV) is a common form of genetic variation underlying animal evolution and phenotypic diversity across a wide range of species. In the mammalian genome, high frequency of CNV differentiation between breeds may be candidates for population-specific selection. However, CNV differentiation, selection and its population genetics have been poorly explored in horses. RESULTS: We investigated the patterns, population variation and gene annotation of CNV using the Axiom® Equine Genotyping Array (670,796 SNPs) from a large cohort of individuals (N = 1755) belonging to eight European horse breeds, varying from draught horses to several warmblood populations. After quality control, 152,640 SNP CNVs (individual markers), 18,800 segment CNVs (consecutive SNP CNVs of same gain/loss state or both) and 939 CNV regions (CNVRs; overlapping segment CNVs by at least 1 bp) compared to the average signal of the reference (Belgian draught horse) were identified. Our analyses showed that Equus caballus chromosome 12 (ECA12) was the most enriched in segment CNV gains and losses (~ 3% average proportion of the genome covered), but the highest number of segment CNVs were detected on ECA1 and ECA20 (regardless of size). The Friesian horses showed private SNP CNV gains (> 20% of the samples) on ECA1 and Exmoor ponies displayed private SNP CNV losses on ECA25 (> 20% of the samples). The Warmblood cluster showed private SNP CNV gains located in ECA9 and Draught cluster showed private SNP CNV losses located in ECA7. The length of the CNVRs ranged from 1 kb to 21.3 Mb. A total of 10,612 genes were annotated within the CNVRs. The PANTHER annotation of these genes showed significantly under- and overrepresented gene ontology biological terms related to cellular processes and immunity (Bonferroni P-value < 0.05). We identified 80 CNVRs overlapping with known QTL for fertility, coat colour, conformation and temperament. We also report 67 novel CNVRs. CONCLUSIONS: This work revealed that CNV patterns, in the genome of some European horse breeds, occurred in specific genomic regions. The results provide support to the hypothesis that high frequency private CNVs residing in genes may potentially be responsible for the diverse phenotypes seen between horse breeds.

Genomic measures of inbreeding in the Norwegian-Swedish Coldblooded Trotter and their associations with known QTL for reproduction and health traits.

BACKGROUND: Since the 1950s, the Norwegian-Swedish Coldblooded trotter (NSCT) has been intensively selected for harness racing performance. As a result, the racing performance of the NSCT has improved remarkably; however, this improved racing performance has also been accompanied by a gradual increase in inbreeding level. Inbreeding in NSCT has historically been monitored by using traditional methods that are based on pedigree analysis, but with recent advancements in genomics, the NSCT industry has shown interest in adopting molecular approaches for the selection and maintenance of this breed. Consequently, the aims of the current study were to estimate genomic-based inbreeding coefficients, i.e. the proportion of runs of homozygosity (ROH), for a sample of NSCT individuals using high-density genotyping array data, and subsequently to compare the resulting rate of genomic-based F (FROH) to that of pedigree-based F (FPED) coefficients within the breed. RESULTS: A total of 566 raced NSCT were available for analyses. Average FROH ranged from 1.78 to 13.95%. Correlations between FROH and FPED were significant (P < 0.001) and ranged from 0.27 to 0.56, with FPED and FROH from 2000 to 2009 increasing by 1.48 and 3.15%, respectively. Comparisons of ROH between individuals yielded 1403 regions that were present in at least 95% of the sampled horses. The average percentage of a single chromosome covered in ROH ranged from 9.84 to 18.82% with chromosome 31 and 18 showing, respectively, the largest and smallest amount of homozygosity. CONCLUSIONS: Genomic inbreeding coefficients were higher than pedigree inbreeding coefficients with both methods showing a gradual increase in inbreeding level in the NSCT breed between 2000 and 2009. Opportunities exist for the NSCT industry to develop programs that provide breeders with easily interpretable feedback on regions of the genome that are suboptimal from the perspective of genetic merit or that are sensitive to inbreeding within the population. The use of molecular data to identify genomic regions that may contribute to inbreeding depression in the NSCT will likely prove to be a valuable tool for the preservation of its genetic diversity in the long term.

A genome-wide association study for harness racing success in the Norwegian-Swedish coldblooded trotter reveals genes for learning and energy metabolism.

BACKGROUND: Although harness racing is of high economic importance to the global equine industry, significant genomic resources have yet to be applied to mapping harness racing success. To identify genomic regions associated with harness racing success, the current study performs genome-wide association analyses with three racing performance traits in the Norwegian-Swedish Coldblooded Trotter using the 670 K Axiom Equine Genotyping Array. RESULTS: Following quality control, 613 horses and 359,635 SNPs were retained for further analysis. After strict Bonferroni correction, nine genome-wide significant SNPs were identified for career earnings. No genome-wide significant SNPs were identified for number of gallops or best km time. However, four suggestive genome-wide significant SNPs were identified for number of gallops, while 19 were identified for best km time. Multiple genes related to intelligence, energy metabolism, and immune function were identified as potential candidate genes for harness racing success. CONCLUSIONS: Apart from the physiological requirements needed for a harness racing horse to be successful, the results of the current study also advocate learning ability and memory as important elements for harness racing success. Further exploration into the mental capacity required for a horse to achieve racing success is likely warranted.

Genetic structure and connectivity analysis in a large domestic livestock meta-population: The case of the Pura Raza Español horses.

The Pura Raza Español (PRE) is an autochthonous Spanish horse population distributed in 65 countries and managed by a single association. Since 1960s, breeding animals have been steadily exported to other countries to establish local subpopulations. We analysed the genetic structure of a PRE horse meta-population (MP) of 215,500 animals from countries with at least 80 active animals (27 countries comprising 77% of the total animals in the complete pedigree). Genotypes from active animals (59% of the total animals in the complete pedigree) were also studied. Genetic analysis of the MP was performed using classical parameters such as inbreeding (F), coancestry, founder contribution, effective number of founders/ancestors (fe/fa) and gene flow between countries. Results showed that the MPF coefficient decreased in the last two decades (1990-2013), indicating an explicit management against F. Founder contribution to genetic variability across countries mainly came from females (73.6%). In general, unbalanced contributions of founders reflected the high loss of genetic diversity along generations (fe/fa as low as 32/19 for the whole MP). Despite this differential contribution, the proportional contribution to the global diversity of each country was similar. The highest within-country coancestry value corresponded to Cuba (0.1509), being the only country with highly inbred individuals (over 12%), and the lowest value corresponded to Spain (0.0574). These results should help to avoid further declines in genetic variability and increases in F levels, especially in small countries like Cuba. In parallel, only nine countries presented descendants in a single population, suggesting a common origin for all countries and/or a substantial exchange of genetic material between populations. Spain and the US showed the highest gene flow rates. These results support the need of a coordinated management strategy, especially promoting the exchange of genetic material to increase the effective population size and maintain the levels of genetic diversity in the PRE horse population.

Age-based partitioning of individual genomic inbreeding levels in Belgian Blue cattle.

BACKGROUND: Inbreeding coefficients can be estimated either from pedigree data or from genomic data, and with genomic data, they are either global or local (when the linkage map is used). Recently, we developed a new hidden Markov model (HMM) that estimates probabilities of homozygosity-by-descent (HBD) at each marker position and automatically partitions autozygosity in multiple age-related classes (based on the length of HBD segments). Our objectives were to: (1) characterize inbreeding with our model in an intensively selected population such as the Belgian Blue Beef (BBB) cattle breed; (2) compare the properties of the model at different marker densities; and (3) compare our model with other methods. RESULTS: When using 600 K single nucleotide polymorphisms (SNPs), the inbreeding coefficient (probability of sampling an HBD locus in an individual) was on average 0.303 (ranging from 0.258 to 0.375). HBD-classes associated to historical ancestors (with small segments ≤ 200 kb) accounted for 21.6% of the genome length (71.4% of the total length of the genome in HBD segments), whereas classes associated to more recent ancestors accounted for only 22.6% of the total length of the genome in HBD segments. However, these recent classes presented more individual variation than more ancient classes. Although inbreeding coefficients obtained with low SNP densities (7 and 32 K) were much lower (0.060 and 0.093), they were highly correlated with those obtained at higher density (r = 0.934 and 0.975, respectively), indicating that they captured most of the individual variation. At higher SNP density, smaller HBD segments are identified and, thus, more past generations can be explored. We observed very high correlations between our estimates and those based on homozygosity (r = 0.95) or on runs-of-homozygosity (r = 0.95). As expected, pedigree-based estimates were mainly correlated with recent HBD-classes (r = 0.56). CONCLUSIONS: Although we observed high levels of autozygosity associated with small HBD segments in BBB cattle, recent inbreeding accounted for most of the individual variation. Recent autozygosity can be captured efficiently with low-density SNP arrays and relatively simple models (e.g., two HBD classes). The HMM framework provides local HBD probabilities that are still useful at lower SNP densities.

Uncovering the impacts of alternative splicing on the proteome with current omics techniques.

The high-throughput sequencing of cellular RNAs has underscored a broad effect of isoform diversification through alternative splicing on the transcriptome. Moreover, the differential production of transcript isoforms from gene loci has been recognized as a critical mechanism in cell differentiation, organismal development, and disease. Yet, the extent of the impact of alternative splicing on protein production and cellular function remains a matter of debate. Multiple experimental and computational approaches have been developed in recent years to address this question. These studies have unveiled how molecular changes at different steps in the RNA processing pathway can lead to differences in protein production and have functional effects. New and emerging experimental technologies open exciting new opportunities to develop new methods to fully establish the connection between messenger RNA expression and protein production and to further investigate how RNA variation impacts the proteome and cell function. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing Translation > Regulation RNA Evolution and Genomics > Computational Analyses of RNA.

A convergent malignant phenotype in B-cell acute lymphoblastic leukemia involving the splicing factor SRRM1.

A significant proportion of infant B-cell acute lymphoblastic leukemia (B-ALL) patients remains with a dismal prognosis due to yet undetermined mechanisms. We performed a comprehensive multicohort analysis of gene expression, gene fusions, and RNA splicing alterations to uncover molecular signatures potentially linked to the observed poor outcome. We identified 87 fusions with significant allele frequency across patients and shared functional impacts, suggesting common mechanisms across fusions. We further identified a gene expression signature that predicts high risk independently of the gene fusion background and includes the upregulation of the splicing factor SRRM1. Experiments in B-ALL cell lines provided further evidence for the role of SRRM1 on cell survival, proliferation, and invasion. Supplementary analysis revealed that SRRM1 potentially modulates splicing events associated with poor outcomes through protein-protein interactions with other splicing factors. Our findings reveal a potential convergent mechanism of aberrant RNA processing that sustains a malignant phenotype independently of the underlying gene fusion and that could potentially complement current clinical strategies in infant B-ALL.

Cannabinoid signaling modulation through JZL184 restores key phenotypes of a mouse model for Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS) is a rare genetic multisystemic disorder characterized by mild-to-moderate intellectual disability and hypersocial phenotype, while the most life-threatening features are cardiovascular abnormalities. Nowadays, there are no pharmacological treatments to directly ameliorate the main traits of WBS. The endocannabinoid system (ECS), given its relevance for both cognitive and cardiovascular function, could be a potential druggable target in this syndrome. We analyzed the components of the ECS in the complete deletion (CD) mouse model of WBS and assessed the impact of its pharmacological modulation in key phenotypes relevant for WBS. CD mice showed the characteristic hypersociable phenotype with no preference for social novelty and poor short-term object-recognition performance. Brain cannabinoid type-1 receptor (CB1R) in CD male mice showed alterations in density and coupling with no detectable change in main endocannabinoids. Endocannabinoid signaling modulation with subchronic (10 days) JZL184, a selective inhibitor of monoacylglycerol lipase, specifically normalized the social and cognitive phenotype of CD mice. Notably, JZL184 treatment improved cardiovascular function and restored gene expression patterns in cardiac tissue. These results reveal the modulation of the ECS as a promising novel therapeutic approach to improve key phenotypic alterations in WBS.

Williams-Beuren syndrome (WBS) is a rare disorder that causes hyper-social behavior, intellectual disability, memory problems, and life-threatening overgrowth of the heart. Behavioral therapies can help improve the cognitive and social aspects of the syndrome and surgery is sometimes used to treat the effects on the heart, although often with limited success. However, there are currently no medications available to treat WBS. The endocannabinoid system ­ which consists of cannabis-like chemical messengers that bind to specific cannabinoid receptor proteins ­ has been shown to influence cognitive and social behaviors, as well as certain functions of the heart. This has led scientists to suspect that the endocannabinoid system may play a role in WBS, and drugs modifying this network of chemical messengers could help treat the rare condition. To investigate, Navarro-Romero, Galera-López et al. studied mice which had the same genetic deletion found in patients with WBS. Similar to humans, the male mice displayed hyper-social behaviors, had memory deficits and enlarged hearts. Navarro-Romero, Galera-López et al. found that these mutant mice also had differences in the function of the receptor protein cannabinoid type-1 (CB1). The genetically modified mice were then treated with an experimental drug called JZL184 that blocks the breakdown of endocannabinoids which bind to the CB1 receptor. This normalized the number and function of receptors in the brains of the WBS mice, and reduced their social and memory symptoms. The treatment also restored the animals' heart cells to a more normal size, improved the function of their heart tissue, and led to lower blood pressure. Further experiments revealed that the drug caused the mutant mice to activate many genes in their heart muscle cells to the same level as normal, healthy mice. These findings suggest that JZL184 or other drugs targeting the endocannabinoid system may help ease the symptoms associated with WBS. More studies are needed to test the drug's effectiveness in humans with this syndrome. Furthermore, the dramatic effect JZL184 has on the heart suggests that it might also help treat high blood pressure or conditions that cause the overgrowth of heart cells.

An investigation into factors influencing basal eye temperature in the domestic horse (Equus caballus) when measured using infrared thermography in field conditions.

Infrared thermography (IRT) is a popular technology used for the detection of thermal changes given its non-invasive nature and lack of direct contact with the individual. Accordingly, the maximal eye temperature (MaxET) measured with IRT has been extensively applied in equine research. However, there is little information available about the potential limitations of the MaxET in field studies. Thus, the aims of this study were to 1) quantify the individual variation of MaxET in field conditions and the effects of individual, breed, body size (height at withers), eye side, sex and age, 2) determine the effects of environment and operator, and 3) explore the relationship between MaxET and rectal temperature (RT) at rest. To accomplish these aims, 791 MaxET measures from 32 horses were collected in Sweden in five different months and five farms over a period of 12 months. There was an effect of individual on IRT (P < .05) and individual MaxET varied from 29.4 to 37.6 °C. IRT was also affected (P < .05) by breed and sex (maximal difference 1.1 °C and 0.3 °C, respectively) but not by eye side, age and height at withers. There were significant effects of month and farm (maximal differences; 2.4 and 2.3 °C, respectively), between outdoor and indoor measurements (0.8 °C) and also between operators (0.2 °C). There were no correlations between MaxET and RT. These results demonstrate that in horses observed at rest in their home environment, MaxET is affected by endogenous (sex and breed) and environmental factors (farm, location and month of the year) and shows no relationship to RT. We strongly suggest that IRT technology should be used with great caution in field studies and only under conditions where these factors can be accurately accounted for.

Benefits and risks of barefoot harness racing in Standardbred trotters.

There is a lack of research on the benefits and risks of shoeing conditions in harness racing. Thus, our objectives were to: (a) investigate whether velocity times (VT; s/km) are affected by racing unshod (N = 76,932 records on 5,247 horses); (b) determine the potential risks of galloping, being penalized, and disqualification when competing unshod (N = 111,755 records on 6,423 horses); and (c) identify additional environmental factors that affect VT and risks. VT was found to be significantly influenced by shoeing condition (e.g., unshod, shod front, shod hind, or fully shod), but also by sex, age, season, track, track condition, start method, start position, distance, and driver-horse performance level (p < 2e-16). The risks of galloping and disqualification were significantly influenced by shoeing condition, sex, age, season, track, start method, start position, or driver-horse performance level (p ≤ .05). Horses racing unshod had 0.7 s/km lower VT than fully shod horses and showed better performance when racing on neutral tracks during the late summer than horses with other shoeing conditions during the same period. However, racing unshod increased the relative risks of galloping and disqualification by 15%-35% in all seasons. Horses shod only on the hind hooves showed better performance than fully shod horses, without higher risks associated with competing unshod.

Ribosome profiling at isoform level reveals evolutionary conserved impacts of differential splicing on the proteome.

The differential production of transcript isoforms from gene loci is a key cellular mechanism. Yet, its impact in protein production remains an open question. Here, we describe ORQAS (ORF quantification pipeline for alternative splicing), a pipeline for the translation quantification of individual transcript isoforms using ribosome-protected mRNA fragments (ribosome profiling). We find evidence of translation for 40-50% of the expressed isoforms in human and mouse, with 53% of the expressed genes having more than one translated isoform in human, and 33% in mouse. Differential splicing analysis revealed that about 40% of the splicing changes at RNA level are concordant with changes in translation. Furthermore, orthologous cassette exons between human and mouse preserve the directionality of the change, and are enriched in microexons in a comparison between glia and glioma. ORQAS leverages ribosome profiling to uncover a widespread and evolutionarily conserved impact of differential splicing on translation, particularly of microexon-containing isoforms.

Stress level effects on sport performance during trotting races in Spanish Trotter Horses.

The stress level is suggested to have a negative effect on horses during equestrian competitions, specifically in trotter racing. The main objectives of this study were to measure stress levels in Spanish Trotter Horse races with a reliable non-invasive method, and determining the threshold level of stress that leads to the best performance results and also shows when the stress perceived by the horse becomes distress. One hundred and thirty individuals were evaluated, measuring their performance (based on racing time per kilometre (TPK)) and their stress (based on eye temperature, assessed with infrared thermography, and heart rate) in different competitions. Eye temperature and heart rate measurements were collected 2h before the race and immediately following the race, and the increases of eye temperature (∆ET) and heart rate were estimated. ∆ET and eye temperature before the race showed significant differences related to the performance level after a GLM analysis. The segmented regression analysis indicated that when the animal was more stressed before the race than just after finishing it (∆ET<0), it showed the poorest competition results, and from the breakpoint (reached at ∆ET=-0.97%), horse's performance started to improve. When comparing eye temperature variables and TPK with a response surface plot, TPK was optimum (77.27s) when the eye temperature before the race and ∆ET reached values of 37.61°C and 7.57%, respectively. Therefore, the stress levels of the horse before the race influence its competition results, and ∆ET during competitions reaching a threshold point is related to an improvement in performance results.