Intramuscular fat is an important factor affecting meat quality and consumer acceptance. Appropriate increases in the intramuscular fat content contribute to the improvement of meat quality, and genetic selection is an effective method to increase the intramuscular fat content in chickens. In this study, chicken lines divergently selected for their intramuscular fat content were used to investigate the mechanisms behind differential intramuscular fat deposition. These results found in this study may contribute to the improvement of meat quality in chickens.p < 0.05) enriched, and nine genes were considered candidate genes for differential IMF deposition. Differences between the lines in the expressions of representative genes involved in the above pathways were detected in 16th generation chickens. This study suggests that genetic selection for increased IMF in the pectoralis major muscle may enhance fatty acid synthesis, transport, and esterification, and reduce triglyceride hydrolysis. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, glycerolipid metabolism, and fatty acid degradation pathway may have contributed to the differences in IMF deposition between the lines. These results contribute to the understanding of the genetic mechanisms behind IMF deposition, and the improvement of chicken meat quality.Intramuscular fat (IMF)—an important factor affecting meat quality—can be appropriately increased by genetic selection. Chicken lines divergently selected for IMF content were used in this study to investigate the mechanisms behind differential IMF deposition. Sixty 15th generation chickens were genotyped using the IASCHICK 55K single nucleotide polymorphism (SNP) chip. After quality control, 59 chickens and 36,893 SNPs were available for subsequent analysis. Population structure assessment indicated that the lines were genetically differentiated. Based on the top 1% paired fixation index values, three pathways were significantly ( Meat quality plays an essential role in consumer acceptance, and intramuscular fat (IMF), deposited within the muscle tissue, is a widely acknowledged indicator of meat quality . NumerouBased on its heritability and adequate variability, it is possible to increase the IMF content through genetic selection. IMF selection programs have been performed in many livestock, including pigs , cattle An IMF selection program using a yellow-feathered chicken breed (Jingxing 100) has been undertaken by our research group since 2000. Our previous research showed that selecting for an increased IMF content resulted in lower shear-force values and improved meat quality in chickens after five generations of selection , therebyThis study was conducted in accordance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology . All experimental protocols were approved by the Science Research Department in charge of animal welfare of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences  (No. IAS2019-21). All birds were raised under the same recommended environmental and nutritional conditions. Basal diets were formulated based on the National Research Council (1994) requirements and the Feeding Standards of Chickens established by the Ministry of Agriculture, Beijing, China (2004).The broiler lines used in this study were derived from the flock at the Institute of Animal Sciences of the Chinese Academy of Agricultural Sciences, which has been divergently selected for IMF content since 2000. The broilers in the two lines originated from the same base population of Jingxing yellow-feathered chickens . In each generation, at least 3 male and 3 female chickens within the same family were slaughtered, and IMF content was measured (described below). The selection was based on the IMF mean within a family. The F Line was selected to increase the IMF, and the C line was a randomly-bred control line, as described by Zhao et al. .In G15, 30 birds from the F line and 30 from the C line were randomly selected at 98 days of age. Blood was collected from the wing veins of the chickens and stored with an anticoagulant until genomic DNA (gDNA) extraction and genotyping.In G16, 8 birds randomly selected per line from the F and C lines. The pectoralis muscle samples were weighed, snap-frozen in liquid nitrogen, and stored at −80 °C until RNA isolation. The remaining pectoralis muscle tissues were removed, weighed, and stored at −20 °C until the measurement of the IMF, triglyceride (TG), phospholipid (PL), and total cholesterol (TCHO) contents.gDNA was extracted from the blood samples from the G15 birds using the phenol-chloroform method. In total, 60 chickens were genotyped using the IASCHICK 55K single nucleotide polymorphism (SNP) chip . Genomic2 values of 0.2 or higher were removed. MDS components were acquired based on the pairwise identity-by-state matrix, and the relative kinship matrix was constructed from these independent SNPs, as described in detail by Sun et al. [https://itol.embl.de) [Multidimensional scaling (MDS) analysis was conducted using PLINK (v1.07) software . Indepenn et al. . Based on et al.  and dispembl.de) .FST [FST values were identified as trait-related SNPs. Genes harboring these SNPs  were selected for pathway and functional enrichment analyses using KOBAS 3.0 [http://kobas.cbi.pku.edu.cn). The significance level for pathway enrichment was set as p < 0.05.The genetic differentiation estimate, known as the pairwise fixation index FST , was calFST  using thOBAS 3.0  . After centrifugation, the supernatant was used for TG, PL, and TCHO measurements. The absorbance for TG and TCHO was measured using a microplate reader at 510 nm, and the absorbance for PL was measured at 600 nm. The assays were performed according to the manufacturers’ instructions.The TG and TCHO contents of the pectoralis major muscle samples were measured using TG and TCHO assay kits , and the PL content was measured using a PL assay kit . Pectoralis major muscle samples (about 2 g) from each chicken were homogenized with absolute ethanol at room temperature and centrifuged . Total RNA was isolated from the pectoralis major muscle sample from each chicken using a total RNA kit , according to the manufacturer’s instructions. After concentration, the purity and integrity were determined, and RNA samples with A260/A280 ratios between 1.8 and 2.0 were used for qRT-PCR.ΔΔCt– method [18S rRNA gene.The primers were designed using Primer Premier 6.0 software, and specificity was determined using BLASTN . The qRTt method , and allt-test. Confidence limits were set at 95%, and p < 0.05 (*) or p < 0.01 (**) was considered significant. Data are presented as the mean ± the standard error (SEM).SPSS version 22.0  was used to test the significance of the differences between the groups using Student’s A total of 60 chickens (30 chickens from each line) and 52,184 SNPs were genotyped using the IASCHICK 55K SNP chip. After quality control steps, the final data included 59 individuals and 36,893 SNPs for subsequent analysis.Population structures were analyzed using MDS and phylogenetic tree analysis. MDS analysis was performed using the genotyped data, and the result was plotted using the first two principal components. This plot showed that the individuals from the two groups were clearly clustered into their respective lines a and thaFST between the two lines was estimated using the 36,893 SNPs, which remained after quality control , including the peroxisome proliferator-activated receptor (PPAR) signaling pathway, glycerolipid metabolism, and fatty acid degradation pathway (ACSL1), PPARα, acyl-CoA dehydrogenase long chain (ACADL), fatty acid binding protein 6 (FABP6), FABP7, phospholipid phosphatase 3 (PLPP3), pancreatic lipase related protein 1 (PNLIPRP1), membrane bound O-acyltransferase domain containing 1(MBOAT1), and aldehyde dehydrogenase 3 family member A2 (ALDH3A2), were enriched in these pathways, and were, therefore, considered important candidate genes for differential IMF deposition in chickens, based on their basic functions.The  control . The mosanalyses . Pathway pathway . Nine gep < 0.01). Given that the TG, PL, and TCHO are the main components of the IMF [p < 0.01), but there were no differences in either the PL or TCHO contents between the two lines (p > 0.05).As shown in PPARα and ALDH3A2 in the pectoralis major muscle tissue were significantly lower in the F line than in the C line (p < 0.05), whereas the expressions of ACSL1, MBOAT1, and PLPP3 were significantly higher than in the C line (p < 0.05 or p < 0.01). In addition, the expressions of several key genes involved in the PPAR signaling pathway were also explored, including PPARβ/δ, fatty acid binding protein 4 (AFABP), malic enzyme 1 (ME1), perilipin 1 (PLIN1), and PPARγ. Relative to the C line, the F line chickens had lower expressions of PPARβ/δ, but higher expressions of AFABP, ME1, PLIN1, and PPARγ (p < 0.05 or p < 0.01).To validate the candidate genes screened in the G15 chickens using the 55K SNP chips, the expression of representative genes in the two lines in G16 was determined using qRT-PCR . The expSelection is an effective way to improve important economic traits in broilers, such as the growth rate and feed conversion ratio. Based on the heritability, chicken lines with divergent IMF contents could be achieved using conventional family-based selection. Indeed, the divergently selected chicken lines used in this study differed significantly in the IMF contents of their breast muscle tissue ,8. GivenFST is a commonly used method to detect selection signatures between lines or breeds in domestic animals [FST values determined in this study, there was moderate genetic differentiation between the lines. Based on the genes found to harbor trait-related SNPs , the classic PPAR signaling pathway, glycerolipid metabolism, and the fatty acid degradation pathway were significantly enriched (p < 0.05), indicating that there were differences in the muscle lipid metabolism process between two the lines. Nine genes  were found to have been enriched in the above pathways. These genes are involved in lipid metabolism and may be important candidate genes for differential IMF deposition. The above results indicated that long-term selection may affect the muscle lipid metabolism process in differentially selected lines.Genotyping data for the G15 chickens were obtained using 55K SNP chips covering 28 autosomal chromosomes. Population structure was assessed using MDS and phylogenetic tree analysis, which confirmed the effectiveness of the selection program and the different selection directions of the two lines. After long-term selection for the IMF content, the chromosome regions that harbor important genes for IMF deposition may exhibit different allele or haplotype frequencies . The FST animals ,27 and cp < 0.01) than in the C line at 98 days of age (breeding age), and that this may have been as a result of differential TG deposition. In addition, qRT-PCR was used to explore the expressions of the representative genes identified in the chickens using the 55k SNP chips. In the pectoralis major muscle tissue of the F line, PPARα was upregulated and ACSL1 was downregulated, relative to the C line. PPARs are classical transcription factors involved in lipid metabolism and include three isotypes  with different tissue distributions and regulatory activities. PPARα is expressed mainly in tissues with a high capacity for fatty acid oxidation. In mammalian liver tissue, PPARα induces fatty acid oxidation through the activities of the mitochondria and peroxisomes, then increases acetyl-CoA production to provide energy [PPARα expression in the F line suggests that lipolytic metabolism in the pectoralis major muscle tissue of these chickens was slower than in the C line chickens. ACSL1 encodes a type of long-chain acyl-CoA synthetase that is involved in both lipid biosynthesis and fatty acid degradation. ACSL1 is upregulated by PPARα ligands in the liver and is elevated by PPARγ agonists in the adipose tissue [ACSL1 overexpression can promote triglyceride accumulation in adipocytes [ACSL1 enhanced the synthesis of triglycerides and resulted in higher IMF contents [ACSL1 expression in the F line than the C line coincided with the greater IMF deposition found in the former. These results indicated that weaker lipolysis and stronger lipogenesis processes may contribute to the higher IMF content found in the F line than the C line. To verify this hypothesis, the expressions of several genes associated with lipolysis and lipogenesis in the PPAR signaling pathway were explored. The expression of PPARβ/δ was downregulated, whereas PPARγ was upregulated in the F line. The effect of PPARβ/δ is similar to those of PPARα, which promotes energy dissipation, but opposite to those of PPARγ, which promotes energy storage [PPARγ in the F line may directly increase PLIN1 expression. Each PLIN family member likely performs a unique function in the regulation of lipid droplet dynamics [PLIN1 was higher in muscles with high IMF contents [AFABP, a negative regulator of mitochondrial fatty acid oxidation [ME1 is correlated with fat deposition in broiler chickens [ME1 in the F line indicated that fatty acid synthase was activated in these chickens. In the PPAR signaling pathway, lipolysis related genes were downregulated (PPARα and PPARβ/δ), and lipogenesis related genes were up regulated  in the F line. These results demonstrated that the F-line chickens exhibited enhanced fatty acid synthesis, transport, and esterification processes, and reduced hydrolysis of triglycerides stored in lipid droplets, which may explain the higher IMF deposition in the F than C line.To confirm this result, chickens in G16 were used to verify the differences in lipid deposition between the two lines. The phenotypic results indicated that the IMF content in the F line was significantly higher , is an intermediate in the synthesis of triglycerides from dietary fats and carbohydrates [PLPP3 gene, is involved in the penultimate step of triglyceride synthesis and has the ability to dephosphorylate PA, thereby generating the triglyceride precursor diacylglycerol [PLPP3 expression in the F line indicated that increased triglyceride synthesis from dietary fats and carbohydrates was taking place in these chickens. In addition, MBOAT1, which was enriched in glycerolipid metabolism pathway, also showed higher expression in the F line. MBOAT1 has been reported to be associated with phosphatidylcholine metabolism [MBOAT1 may, thus, also be associated with differences in IMF deposition between the two lines.In addition to the PPAR pathway, several other pathways were also involved in the differential IMF deposition between the two lines. xidation . It has  embryos . The exphydrates . LPP3, wglycerol . Upregultabolism  and largtabolism . An upreFST is an effective method to detect selection signatures. However, it is unavoidable to yield false signatures depending on a single approach. Na et al. [FST and hapFLK to explore selection signatures for chicken abdominal fat deposition, while there was no overlap between two methods. Therefore, the relative gene expression was used to verify the reliability of the results in this study. However, by the limitation of sample size and chip density, it is necessary to explore mechanisms of differential IMF deposition by enlarging sample size and sequencing depth in further studies.The possible mechanisms of differential IMF deposition by selection have been revealed in this study. However, there were still some limitations to this research worth discussing. There was no doubt that a et al.  tried toDivergent selection for the IMF content led to differential IMF deposition in the pectoralis major muscle tissue between the two lines. Genetic selection for increased IMF in chickens’ pectoralis muscle tissue may be achieved through the upregulation of the expression of genes involved in fatty acid synthesis, transport, and esterification, as well as the downregulation of the expression of genes involved in lipolysis in the PPAR signaling pathway. In addition, several other pathways also contribute to this process, including glycerolipid metabolism and the fatty acid degradation pathway. The regulatory networks involved in these pathways and the expressions of these genes may be responsible for the increased IMF content in the F line chickens after multiple generations of selection.


PRKDC and PUM2, with a potential prognostic value, were never previously associated with STUMP. The methylation data appears to be very promising, especially with regards to the divergent profile found in the sample that relapsed, characterized by an overall CGI hypomethylation. Finally, the Gene Ontology analysis highlighted some cancer genes that could play a pivotal role in the unexpected aggressive behavior that can be found in some of these tumors. These genes could prove to be prognostic markers in the future.Uterine smooth muscle tumors of uncertain malignant potential (STUMPs) represent a heterogeneous group of tumors that cannot be histologically diagnosed as unequivocally benign or malignant. For this reason, many authors are working to obtain a better definition of diagnostic and prognostic criteria. In this work, we analyzed the genomic and epigenomic profile of uterine smooth muscle tumors (USMTs) in order to find similarities and differences between STUMPs, leiomyosarcomas (LMSs) and leiomyomas (LMs), and possibly identify prognostic factors in this group of tumors. Array-CGH data on 23 USMTs demonstrated the presence of a more similar genomic profile between STUMPs and LMSs. Some genes, such as  According to the 2014 WHO, uterine smooth muscle tumors (USMTs) consist of a group of histologically heterogeneous and clinically diverse diseases ranging from malignant leiomyosarcoma (LMS) to benign leiomyoma (LM). In between the aforementioned, lies a heterogeneous group of rare mesenchymal tumors that cannot be clearly categorized as benign or malignant lesions. Uterine smooth muscle tumors of uncertain malignant potential (STUMPs) do not meet benign variant or true malignancy criteria, due to their variability in histologic appearance, immunohistochemical profile and clinical outcome [Although the majority of patients with uterine STUMPs have good outcomes, unexpected aggressive behavior can be found in some of the tumors . BecauseIn this work, we analyzed the genomic profile of 23 USMTs by array-comparative genomic hybridization (array-CGH). In four samples the methylation profile was also investigated in order to find similarities and differences between STUMPs, LMSs and LMs, and possibly identify prognostic factors in this group of tumors.We performed an array-CGH analysis on 23 USMTs: 3 LMs, 14 STUMPs, 5 LMSs and 1 undifferentiated sarcoma (US), derived from a total of 20 patients who underwent first surgery between 1996 and 2013. The histological characteristics, the age at diagnosis and the type of surgery are listed in Among the 14 STUMPs, 11/14 were primary tumors, of which four  subsequently relapsed in either another STUMP or undifferentiated sarcoma (US) or LMS. For three patients it was possible to analyze both the primary STUMP and its recurrence . Primary STUMPs of patients 8 and 9 and relapsed tumor of patient 7 were not available. Among the LMSs, three were primary , while two were relapsed tumors (LMS1 and LMS5).STUMP samples revealed a wide range of copy number alterations (CNAs), from 17 to 365, with a median value of 86.5 .Two samples (STUMP3 and STUMP4) showed an exceptional number of CNAs (365 and 256 respectively). LMSs’ total number of CNAs ranged from 126 to 262, with a median value of 199. The only US sample analyzed showed the least number of CNAs compared to other sarcomas. Finally, LMs displayed an unexpected number of CNAs, ranging from 110 to 268, with a median value of 123. No statistically significant differences for the total number of CNAs were observed among the groups. A CNAs plot by chromosome for each patient is displayed in Interestingly, combining these data with the follow-up status, we found that primary STUMPs and primary LMS derived from patients with a worst prognosis  displayed a higher number of CNAs compared to patients’ samples with a good prognosis  A. As shop = 0.0004; p = 0.01; LMs vs. not relapsing STUMPs, Tukey test p = 0.0008, data not shown). No other differences were observed between the two groups of STUMPs or between STUMPs and LMSs.ANOVA analysis performed on CNAs’ distribution within chromosomes in LMs, STUMPs and LMSs revealed a statistically significant difference only in chromosome 17 . The majority was exclusive; few were shared between two samples. Moreover, known cancer-associated genes were not found (data not shown).PRKDC , MLL3 , ROCK2 , CTSB  and STAT2   . InteresSTAG1 , RFC4 , CCRN4L and ELF2  , including gain/amplification of the regions encompassing samples) .BCL2 gene in primitive STUMPs and their corresponding relapsed tumors have been previously reported [BCL2 data was performed, revealing copy number gain or amplification in 13 samples: one leiomyoma (LM2), four primary STUMPs , two relapsing STUMPs (STUMP3 and STUMP4), one relapsed STUMP (STUMP6), three primary leiomyosarcomas  and two relapsed LMSs (LMS1 and LMS5)  .p < 0.01 Student’s t test). In particular, the percentage of methylated CGIs for all chromosomes in STUMP4 was significantly lower  compared to the one observed in the other three samples, which presented similar percentages, around 50%, of methylated and unmethylated CGIs  and two LMSs (LMS1 and LMS2). STUMP8 and LMS2 were primary lesions that had not yet relapsed, while LMS1 was the relapse of STUMP4. The array platform used in this study covers 27,800 CpG Island (CGIs) of the human genome and all the data (percentages and frequencies) are referred to the total number of CGIs included in the array. We calculated the percentages of unmethylated/methylated/undetermined CGIs and we observed that STUMP4, the primary lesion that relapsed, had a divergent methylation pattern compared to the other three samples (ted CGIs B.In order to perform a Gene Ontology (GO) analysis, we set up two lists of genes on the basis of the promoter methylation status. The first list included the 667 genes which were methylated, while the second one the 2220 genes that resulted unmethylated in all samples. Cancer-related GO terms were grouped in different functional categories, as previously described [p-value = 1.92 × 10−35) and 99 genes  from the unmethylated and methylated gene lists respectively. We found that gene associated GOterms such as ‘transcription and gene expression’, ‘cell cycle’ and ‘cell signalling’ were detected in both the unmethylated and methylated genes’ lists. Conversely, GOterms such as ‘transport’ and ‘DNA repair and chromatin remodeling’ and ‘development and morphogenesis’ and ‘cell death and apoptosis’ were found almost exclusively in the unmethylated or methylated group of genes, respectively.We subsequently refined our analysis, limiting the GOgroups to ‘biological process’ aspects . The most significant GO-groups included 336 genes  were mainly involved in ‘transcription and gene expression’, ‘metabolism’, ‘cell signaling’, ‘cell cycle’ and ‘cell death and apoptosis’ categories, as verified in the previous analysis. Surprisingly, only four of the methylated genes were included in the most significant GO-group  and were involved in ‘cytoskeleton organization’. Furthermore, statistical analysis (chi-square test) revealed a significant difference for some categories between STUMP4 and the other samples . In conclusion, we may assume that Ras protein signal transduction, Wnt signalling pathway and NFKbeta cascade were more involved in the primary lesion than in its respective recurrence, being more represented in STUMP4 unmethylated promoters . In order to perform the Gene Ontology analysis, we established two lists of genes. The first list included 628 genes, which were found methylated in STUMP4 and unmethylated in LMS1. Conversely, in the second list 2972 genes were reported resultingunmethylated in STUMP4 and methylated in LMS1. As expected, ‘metabolism’ and ‘transcription and gene expression’ were the most represented biological functions in both samples, but no statistically significant differences were identified (data not shown). However, the percentage of unmethylated promoter CGIs in the ‘Cell signalling’ category differed in the two samples: STUMP4 displayed 3.1% of unmethylated genes (92/2972) compared to 9.2% (58/628) of LMS1 . This gene is a key component of the non-homologous end joining pathway for DNA repair and its overexpression and/or copy number gain have been observed in several cancer types, such as lung cancer, liver cancer and colorectal cancer, and are associated with more advanced tumor grade and poor survival [It is already known that copy number gains or amplifications are associated with protein overexpression in cancer. Eight samples (four STUMPs and four LMSs) shared gain or amplification of survival ,9. Recensurvival  and in bsurvival . In our MLL3 (Mixed Lineage Leukemia 3) copy number gain or amplification was shared among seven samples (three STUMPs and four LMSs). Intriguingly, two STUMPs subsequently relapsed and the third was a relapsed STUMP. Mutations of this gene were reported in different tumors [t tumors , but notROCK2 and CTSB gains or amplifications were shared among six samples (both STUMPs and LMSs). ROCK2 (Rho associated coiled-coil containing protein kinase 2) upregulation has been described in human glioblastoma CSCs [ROCK2 expression level has been associated with worse prognosis in osteosarcoma tissues [CTSB (cathepsin-B) overexpression has been identified in several tumors with different effects on patient survival [oma CSCs , in gastoma CSCs , as welloma CSCs . Moreove tissues . CTSB . Its amplification and overexpression in breast and ovarian cancer cell lines has been previously demonstrated [We refined the analysis comparing only the relapsing STUMPs with LMSs, and we found some shared CNAs. In particular, we observed a copy number gain in four samples (one STUMP and three LMSs) involving nstrated .RFC4 (Replication Factor C Subunit 4) gain with three LMSs. Overexpression of RFC4 commonly occurs in colorectal cancer and higher levels of RFC4 protein expression correlate with a worse overall survival [The same STUMP sample (the only relapsed in a LMS) shared survival . Moreovesurvival .ELF2 (E74 Like ETS Transcription Factor 2) copy number gain was shared among only three samples, but its role in tumorigenesis has been well described. In fact, overexpression of ELF2 enhanced tumor cell proliferation [Finally, feration  in nasopferation .BCL2 gene in primitive STUMPs and their corresponding relapsed tumors [BCL2 amplification was observed in different samples (eight STUMPs and five LMSs), not only the ones that relapsed. It is therefore clear that an increase of analyzed cases is crucial for the validation of new targets. In this context, we collected and examined 14 STUMPs, a significant number for this rare type of tumor.We previously identified the amplification of d tumors . This obEpigenomic modifications, such as DNA methylation, are an integral part of the molecular determinants, contributing to malignancy . For theIn cancer, the role of promoter hypermethylation in silencing tumor suppressor genes and its impact on tumor initiation, progression and prognosis are well known. Even though DNA hypomethylation was the first epigenetic disruption in cancer , it tookOur data showed a threefold of genes with unmethylated promoter CGIs compared to the methylated ones, considering all four samples. As expected, no statistically significant differences were highlighted by the Gene Ontology analysis comparing the four samples. Conversely, significant differences for several categories emerged from the isolation of the sample with a different profile of methylation (STUMP4), and the comparison with the other three. This analysis highlighted specific signaling pathways that might be preferentially activated in this tumor. The same conclusion emerged thanks to the comparison of the primary sample with its recurrence, outlining a distinctive behavior of the sample with the worst prognosis.In conclusion, our data demonstrated a more similar genomic profile between STUMPs and LMSs with some genes with a potential prognostic value. Despite the lack of an NGS-based point mutations investigation, which would have given a complete picture of the mutation burden (the landscape), and the number of samples being too small to draw conclusions, the Gene Ontology analysis highlighted some cancer genes that could play a pivotal role in the unexpected aggressive behavior in some of these tumors. These genes could prove to be prognostic markers in the future. Finally, the methylation data appears to be very promising, especially with regards to the divergent profile found in the sample that relapsed, despite the analysis being restricted to only four samples.A total of 23 uterine smooth muscle tumors: 3 leiomyomas, 14 STUMPs, 5 leiomyosarcomas and 1 undifferentiated sarcoma, were collected from a total of 20 patients . ComitatIn our case, we based the diagnosis of STUMP following the WHO indication: a uterine SMT that cannot be diagnosed unequivocally as malignant  or cleaAs far as we are concerned, all the specimens defined as STUMP had <10 mitosis/10 HPFs, but were not classified as benign because of the presence of tumor necrosis even without atypia or additional histological parameters that may predict adverse outcome, as reported by Gupta et al.   sections of 1-µm thickness. The entire pre-treatment process of deparaffinization, rehydration and epitope retrieval was performed using PT LINK . Then the sections were placed into the Autostainer Link 48 with the EnVision FLEX visualization system.Estrogen receptor (ER), progesterone receptor (PgR), p16, p53, Ki67 (for mitotic activity) and other markers were evaluated . For interpretation of immunohistochemical staining, the system proposed by Ip et al.  was adop® spin Tissue Mini Kit  using 10 µm slices from formaline-fixed paraffin embedded biopsies. All DNAs were quantified by Nanodrop-1000  and DNA integrity was measured through agarose gel electrophoresis; generally all FFPE DNA appeared partially fragmented.DNA extraction from Formalin-Fixed Paraffin-Embedded (FFPE) tissues was performed by InvisorbSample preparation, slide hybridization and analysis were performed using SurePrint G3 Human CGH Microarray 8 × 60 K  based on the UCSC Genome Browser hg18, NCBI build 36.1, March 2006 with 41Kb overall median probe spacing (33 Kb in RefSeq genes), according to the manufacturer’s instructions for FFPE samples. A control female DNA from a healthy female blood donor was used as sex-match reference. This control DNA was validated by testing on CGH microarray using as references available commercial European Male DNA NA-1289 and European Female DNA NA-12878  and by self-hybridization. All the samples were heat fragmented at 99 °C for 10 to 20 min and 250 ng of fragmented DNA per sample were directly labelled by Genomic DNA ULS-labelling Kit (Agilent Technologies) according to the protocol. ULS-labelling kit does not copy or amplify the input DNA, so the yield after the labelling is the same as the input DNA. The arrays were scanned at 2 µm resolution using the Agilent microarray scanner and analyzed using Feature Extraction v10.7 and Agilent Genomic Workbench v5.0 softwares . The Aberration Detection Method 2 (ADM-2) algorithm was used to compute and assist the identification of aberrations in a given sample (threshold = 5.0), assigning a statistical score based on the average quality weighted log ratio (DLRS) of the sample and reference channels. ADM2 threshold filter selected at least three contiguous probes with an average absolute value of the log2ratio change of 0.60 across the aberration in order to define gains and losses. Additional abnormalities were manually noted when detected via visual inspection of the experimental data points for each chromosome. The estimated percentage of mosaicism was calculated using the formula determined by Cheung SW et al. .Methylated DNA immunoprecipitation and chip hybridization were performed following the guidelines of Agilent Microarray Analysis of Methylated DNA Immunoprecipitation Protocol  as previously described . Brieflyhttp://gostat.wehi.edu.au/ accessed on 21 May 2020 [The Gene Ontology analysis was performed using the GOstat software (May 2020 ) in ordeMay 2020 . AnalyzeGOstat analysis was also performed narrowing the GO hierarchy through the use of ‘biological_function’ as a keyword.


This study aimed to examine the choroidal thickness profiles in visually normal Australian Indigenous children, given the important role of the choroid in refractive error and a range of ocular diseases.Choroidal thickness was assessed across the central 5 mm macular region using enhanced depth imaging spectral domain optical coherence tomography, in 250 children enrolled in an elementary school and a secondary school in rural Queensland, Australia. One hundred (40%) of these children identified as Indigenous Australians.P = 0.03). Subfoveal choroidal thickness was also significantly associated with age , and axial length . A significantly thicker choroid in Indigenous children was also found in analyses across the central 5 mm macular region (P = 0.008). A significant interaction between Indigenous status and meridian was observed (P = 0.007) with the largest differences between Indigenous and non-Indigenous children being in the nasal and inferonasal meridians.The subfoveal choroid was significantly thicker in Indigenous children (mean 369 ± 75 µm), compared to non-Indigenous children (355 ± 73 µm; This study establishes the normative characteristics of macular choroidal thickness in Indigenous Australian children and demonstrates a significantly thicker choroid compared to non-Indigenous children from the same geographic region. These results may have implications for our understanding of factors predisposing or protecting Australian Indigenous people from a range of conditions associated with choroidal thickness.The significantly thicker choroid in Australian Indigenous children should be considered in clinical diagnoses and management of conditions associated with choroidal changes. Knowledge of the normal characteristics of the choroidal structure in the human eye is important given the role of the choroid in normal retinal function,–––In recent decades, in vivo choroidal changes in the human eye associated with refractive error have been well documented. Cross-sectional studies of both adults–Choroidal structural changes have also been shown to be associated with a range of different ocular diseases.,–,,,,,,A range of demographic factors are also known to influence the normal choroidal thickness. In adults, choroidal thickness has been reported to be greater in males than females.,,,Only a small number of studies to date have examined whether ethnicity influences choroidal thickness.,In Australia, Aboriginal and/or Torres Strait Islander people comprise approximately 3% of the population.,Although only a small number of studies have provided detailed information regarding refractive error in Australian Indigenous populations, studies of both adults and children report a very low prevalence of myopia compared to other populations.In this study, we aimed to characterize the normal topographical characteristics of choroidal thickness in Indigenous Australian children. Given the strong association between choroidal thickness with a range of eye diseases and refractive error, an improved understanding of the normal choroidal characteristics of Indigenous children is important for future diagnosis of eye disease and better understanding of eye growth and refractive error in this population.n = 242) and a high school (n = 347) in rural South East Queensland, Australia, were invited to participate in this research. These schools were selected due to their high enrollment of Indigenous children. Two hundred fifty children aged between 4 and 18 years (mean age 11.7 ± 3.4 years) who agreed to participate in this study and had normal vision and ocular health were included in the analyses presented in this paper. Approval from the Queensland University of Technology human research ethics committee and from the Queensland Government Department of Education was provided before commencement of the study and written parental consent and child assent was provided by all participants. If children were unable to provide written assent, then verbal assent was provided. All children were treated according to the tenets of the Declaration of Helsinki. Indigenous status  was established based upon a questionnaire completed by each child's parent or guardian. Both Indigenous and non-Indigenous children were enrolled in the study.All children enrolled at a primary school , ocular biometry (using the Lenstar LS900 instrument), and spectral domain OCT imaging. To ensure that choroidal thickness measures were not confounded by changes due to the effects of cycloplegic drugs on the choroid,P < 0.001). There were no other statistically significant differences between the Indigenous and non-Indigenous children in terms of age, gender balance, astigmatic refractive error, or axial length .OCT images of the right eye only were analyzed from those children with best corrected vision of 20/32 or better, no signs of strabismus, no history of  or evidence of ocular disease, and no history of  systemic disease that might affect the choroid or refractive status were included in the analyses. The 250 children included in the final analyses, comprised 100 Indigenous children and 150 non-Indigenous children . These cOCT imaging was performed using the Heidelberg Spectralis Instrument , which has previously been shown to provide highly repeatable measures of choroidal thickness in children.Following image capture, all OCT scans were exported and analyzed using custom written software. Initially all OCT images were analyzed using automated algorithms based upon deep learning methods, to segment the outer boundary of the retinal pigment epithelium (RPE), and the inner boundary of the chorioscleral interface (CSI).n = 8; 5 Indigenous children and 3 non-Indigenous children) were unable to maintain stable fixation for sufficiently long to capture all six scan lines, and therefore their data were only included in the subfoveal analyses.The choroidal thickness data from each child were then analyzed to calculate the subfoveal choroidal thickness . The choroidal thickness across the central 5 mm diameter surrounding the fovea in each scan was analyzed to determine the average foveal , parafoveal (average thickness from 1 mm to 3 mm diameter), and perifoveal (average thickness from 3 mm to 5 mm diameter) choroidal thickness, over 8 different meridians . A small number of participants  was used to examine the effect of Indigenous status upon subfoveal choroidal thickness, including age and axial length as covariates. To assess the central 5 mm macular choroidal thickness, a repeated measures ANOVA was conducted with two within-subject factors  and the between-subject factor of Indigenous status, with axial length and age included as covariates.1,246 = 4.29, P = 0.03; β = + 7.6 µm/year, r2 = 0.105, F1,246 = 30.15, P < 0.001; β = −19.9 µm/mm, r2 = 0.030, F1,246 = 9.6, P = 0.002) was also found (P > 0.05). There was no significant effect of gender (P > 0.05).The mean subfoveal choroidal thickness of all children was 361 ± 74 µm (interquartile range = 308 to 409 µm) and was significantly thicker in Indigenous children  than non-Indigenous children  and axial length  were also observed.Across the central 5 mm, significant topographical variations in thickness were found in both Indigenous and non-Indigenous children , Table 21,238 = 7.1, P = 0.008) in Indigenous children compared to non-Indigenous children . A significant interaction between meridian and Indigenous status was also observed , with the largest differences between Indigenous and non-Indigenous children observed in the nasal  and inferior nasal  meridians, and the smallest difference in the superior meridian  indicating that the difference in thickness between Indigenous and non-Indigenous children was similar between the foveal, parafoveal and perifoveal zones. The average macular choroidal thickness was significantly greater , Australian Indigenous children had a significantly thicker macular choroid. Previous reports of mean subfoveal choroidal thickness in populations of primarily non-myopic children from a variety of different countries,,,,,,In this primarily non-myopic population of children with a relatively wide range of ages (from 4 to 18 years), we found a significant increase in choroidal thickness associated with increasing age, with a 7.6 µm increase in subfoveal choroidal thickness per year observed during childhood development. These findings are consistent with previous cross-sectional,,,Across the entire cohort, significant topographic variations were also observed in choroidal thickness across the macular region, with central regions generally thicker than more peripheral regions, temporal regions thicker than nasal, and superior thicker than inferior. This topographical distribution of macular choroidal thickness is broadly similar to previous reports in children,,,,Studies of normal eye growthOur findings of a significantly increased choroidal thickness in Australian Indigenous children, may also provide some insights into the potential factors predisposing or protecting Australian Indigenous people from eye disease later in life. Studies indicate that Indigenous Australian adults exhibit a significantly higher rate of diabetic macular oedema compared to non-Indigenous Australians with diabetes.A limitation of this study is the cross-sectional study design, which limits the conclusions that can be drawn regarding the time course of variations in choroidal thickness occurring with age and the development of refractive error. Further longitudinal studies will help to better understand these changes and their relationship with eye growth. Although recruiting all participants living in the same rural location reduces the potential for environmental differences between groups to influence comparisons, it does limit the generalizations that can be made to children living in other locations. Further work exploring choroidal thickness of children living in urban as well as more remote regions will expand our understanding of choroidal thickness in Australian Indigenous children.In conclusion, this study provides the first assessment of choroidal thickness in Australian Indigenous children and demonstrates a significantly greater choroidal thickness in this population. These findings help to establish the normative characteristics of the choroid in Indigenous Australians, which may assist in the future diagnosis and monitoring of choroidal abnormalities. Furthermore, although additional research is required to characterize the normal choroidal thickness of Indigenous Australians across a wider range of ages, our findings may have implications for the potential risk for the development of a range of conditions associated with choroidal changes later in life among Indigenous Australians.


Ophraella communa, a promising BCA of common ragweed, recently and accidentally introduced into Europe. A total 33.98 Gb of raw DNA sequences, representing ∼43-fold coverage, were obtained using the PacBio SMRT-Cell sequencing approach. Among the five different assemblers tested, the SMARTdenovo assembly displaying the best scores was then corrected with Illumina short reads. A final genome of 774 Mb containing 7,003 scaffolds was obtained. The reliability of the final assembly was then assessed by benchmarking universal single-copy orthologous genes  and by remapping tests of Illumina short reads (average of 98.6 ± 0.7% without filtering). The number of protein-coding genes of 75,642, representing 82% of the published antennal transcriptome, and the phylogenetic analyses based on 825 orthologous genes placing O. communa in the monophyletic group of Chrysomelidae, confirm the relevance of our genome assembly. Overall, the genome provides a valuable resource for studying potential risks and benefits of this BCA facing environmental novelties.Rapid evolution of weed biological control agents (BCAs) to new biotic and abiotic conditions is poorly understood and so far only little considered both in pre-release and post-release studies, despite potential major negative or positive implications for risks of nontargeted attacks or for colonizing yet unsuitable habitats, respectively. Provision of genetic resources, such as assembled and annotated genomes, is essential to assess potential adaptive processes by identifying underlying genetic mechanisms. Here, we provide the first sequenced genome of a phytophagous insect used as a BCA, that is, the leaf beetle  Rapid evolution of weed biological control agents (hereafter: BCAs) to both biotic and abiotic environments is poorly understood and so far only little considered both in pre-release and post-release studies of weed biological control programs , nor in Leptinotarsa decemlineata, highlighted a high rate of transposable elements (TEs) compared with other beetle species, high levels of nucleotide diversity and recent expansion of introduced populations that might explain why and how this pest species rapidly evolves on novel host plants, under climate change and/or to overcome management practices (Ophraella communa LeSage 1986 (Coleoptera: Chrysomelidae) for the control of common ragweed, Ambrosia artemisiifolia.Since the emergence of high throughput sequencing methods, population genomic approaches have gained in popularity for studying rapid evolutionary changes in introduced species . This haractices . Unlike A. artemisiifolia L. (Asteraceae), is one of the most prominent plant invaders worldwide, causing economic losses due to severe impacts on human health resulting from its huge amount of highly allergenic pollen and as an important and hard-to-control agricultural crop weed . Its presence on this important economical crop, on which the leaf beetle can even complete its life cycle, was a reason to reject O. communa as BCA for Australia  tribe , includiustralia . The risustralia , an unceO. communa using sequencing of long-reads. We used several assemblers to get the best genome assembly and then assessed its quality by checking for bacterial contamination and for the presence of conserved insect representative genes. In addition, we evaluated the reliability of this genome assembly to perform population genomic studies by remapping tests, performed a preliminary annotation of functional proteins, and replace the species in the phylogeny of Polyphaga Emery, 1886 (Coleoptera). So far, mitochondrial sequences were the single genetic resources available  and reared under random mating, in the quarantine facilities at the University of Fribourg (Switzerland). After 9 months, corresponding to ∼6 generations, four alive adults  were selected for genomic DNA (gDNA) isolation and preserved in Silica gel. The entire individuals were first dried at 70 °C during 1 h and then pooled before grinding to increase the gDNA content at the end of the DNA isolation step. We assumed that the individuals selected were more genetically similar compared with the individuals of the original population, although this similarity is not expected to be high after six generations of rearing under random mating. This expectation is dependent to the homozygosity rate in the original population and the reproductive skew in the insect species. Under random-mating, the gain of homozygosity from the original population is expected to be ∼6% after six generations of rearing. However, evidences for assortative mating have been reported in this chrysomelid species under controlled conditions  according to the manufacturer’s protocol. The resulting library was size selected on a BluePippin system  for molecules larger than15 kilobases (kb). The final library was then sequenced on a PacBio Sequel II instrument (Pacific Biosciences) using five SMRT cells v3, to reach an expectation of ∼50-fold coverage for a genome size estimated at 600 Mb .https://github.com/pacificbiosciences/genomicconsensus, last accessed May 27, 2020) to correct sequence data into consensus sequences with long reads; for the last polishing, we employed Pilon . For this, we used bwa v0.7.17  to map sThe gene prediction was performed with Augustus v3.2.3  using pahttp://www.girinst.org/repbase/, last accessed May 27, 2020) to determine the overall TEs content in the genome assembly using RepeatMasker , selected as an outgroup for the phylogenetic tree. The 825 single-copy orthologous proteins were retrieved for every species using BUSCO. The protein sequences were first aligned with Muscle v3.8.1551 (https://github.com/nylander/catfasta2phyml). The maximum likelihood phylogeny was then estimated using RAxML v8.2.12  absence of bacterial contamination in our genome assembly.The BUSCO assessment indicated an excellent representation of Insect genes, with 96.0% of the 1,658 single-copy orthologs identified as completed , whereas only 1.4% and 2.6% were considered as fragmented and missing, respectively, in the assembly. These percentages are similar to the three other chrysomelid genomes A.Disregarding the population level, the remapping tests showed high percentages of mapping B, suggesO. communa. This number represents 82% of the number of unigenes  predicted by Overall, 75,642 protein-coding genes were predicted in the genome assembly of A high fraction of TEs (58.2%, details per category are provided in O. communa form a monophyletic group with the three other chrysomelid species included in the phylogenetic analysis (The maximum likelihood tree obtained with 825 single-copy orthologous proteins shows that analysis D. The foanalysis .O. communa, a promising biocontrol candidate against common ragweed, one of the world’s most noxious plant invaders. The various quality assessments showed that the genome assembly described in this study is well suited to be used as a reference genome for O. communa and probably also for the other thirteen Ophraella species. In addition to the transcriptome recently published, the preliminary annotation carried out on the present genome assembly will supply the first insights into molecular mechanisms involved in rapid evolution. Overall, this new genome provides a valuable resource to determine potential risks and benefits of weed BCA facing environmental novelties.We provide the first genome assembly of a phytophagous insect used as a BCA, that is, the leaf beetle evaa102_Supplementary_DataClick here for additional data file.


To overcome or minimize this, in the present work a flexible petroleum-derived polyester, namely poly(ε-caprolactone), was used to obtain PHBH/PCL blends with different compositions (from 0 to 40 PCL wt %) using extrusion followed by injection moulding. The thermal analysis of the binary blends was studied by means of differential scanning calorimetry (DSC) and thermogravimetry (TGA). Both TGA and DSC revealed immiscibility between PHBH and PCL. Mechanical dynamic thermal analysis (DMTA) allowed a precise determination of the glass transition temperatures (Tg) as a function of the blend composition. By means of field emission scanning electron microscopy (FESEM), an internal structure formed by two phases was observed, with a PHBH-rich matrix phase and a finely dispersed PCL-rich phase. These results confirmed the immiscibility between these two biopolymers. However, the mechanical properties obtained through tensile and Charpy tests, indicated that the addition of PCL to PHBH considerably improved toughness. PHBH/PCL blends containing 40 PCL wt % offered an impact resistance double that of neat PHBH. PCL addition also contributed to a decrease in brittleness and an improvement in toughness and some other ductile properties. As expected, an increase in ductile properties resulted in a decrease in some mechanical resistant properties, e.g., the modulus and the strength  decreased with increasing wt % PCL in PHBH/PCL blends.Polyhydroxyalkanoates (PHAs) represent a promising group of bacterial polyesters for new applications. Poly(3-hydroxybutyrate- Nowadays, awareness of environmental protection, sustainable development, and the use of renewable energies has become a priority for our society. The high volume of wastes generated that are harmful for the environment, oceans, ecosystems, and so on, has become a major problem to be solved. Furthermore, the waste generated in a consumer society, such as the present one, comes mainly from the packaging sector. This need has favoured the development of new environmentally friendly materials . For thico-terephthalate)—PBAT, and PCL, among others. However, biopolyesters also include bacterial polyesters (PHAs) and some starch-derived polymers such as PLA. The main advantage of polyesters  is that they can undergo biodegradation , through the action of microorganisms. This makes composting an important and simple sustainable option for the management of these wastes  at 25 °C vs.  at −40 °C. At higher temperatures, E’ tended to have very low values (close to 0 MPa) for blends with high PCL wt %. This effect was because PCL melted at about 60 °C, and once this temperature was overpassed, PCL changed from a rubber-like state to a melt state with extremely low elastic properties. Nevertheless, in blends with 10 and 20 wt % PCL  the effect was less pronounced so that at 60 °C, E’ was 178.5 and 142.0 MPa respectively. These values are typical of a rubber-like material and decrease as the wt % PCL increases.The values of reported ,45. At 2Tg of PCL  MPa with 20 PCL wt % and 40 PCL wt %, respectively. This decrease in the resistance parameters was due to the two-phase structure of the blends, resulting from the lack of (or very poor) miscibility, as mentioned above. The immiscibility between PHBH and PCL forms a dispersed PCL phase that interrupts the continuity of the PHBH-rich matrix phase, making the stress transfer difficult and, subsequently, decreasing the Et and σt of the PHBH/PCL blends with increasing PCL wt %.L blends . It shoub(%), the effect was opposite and very positive. The increase in εb(%) in the PHBH/PCL blends also increased ductility with PCL wt %. PHBH is a rather fragile polymer  with only 13.9% elongation at break. Some researchers attributed this fragility to the secondary crystallization or aging on PHBH, which reduced the amorphous fraction [b(%) increased up to 67.8% . Even more, the εb(%) for the blend with the highest PCL wt % considered in this study, i.e., 40 wt %, showed an εb(%) of 461% (this was 33 times higher than neat PHBH).With respect to the elongation at break, εfraction ,19. WithEf). Neat PHBH had an Ef value of 1029 MPa, and this decreased progressively to 801.7 MPa for the PHBH/PCL blend containing 40 PCL wt %. In this case, the decrease in the flexural strength (σf) was not so pronounced as that observed in tensile conditions. In addition, the Shore D hardness, as it is a mechanical resistant property, it followed the same tendency as that observed for both modulus and strength. PHBH showed a Shore D of 61, and the addition of the flexible PCL to PHBH decreased the Shore D values down to 55 for the blend with 40 PCL wt %. Despite this, all Shore D values were close to 58 with very slight variations.−2; with the addition of only 10 PCL wt %, it was increased to 8.7 kJ·m−2 (which represented a % increase of 56%). This same trend was proportionally maintained as the PCL wt % increased. It is worth noting that the impact energy for the PHBH/PCL blend with 40 PCL wt % was around 10.5 kJ·m−2, almost twice as much as neat PHBH. These results were consistent with the above-mentioned decrease in the intrinsic brittleness of neat PHBH by blending with PCL [Another important property of polymers is toughness. with PCL .To check that this dispersed phase corresponded to PCL present in the binary blends, a selective PCL extraction with acetone was performed for 24 h . Figure The processing and obtaining of PHBH/PCL binary blends allowed a new environmentally friendly material to be obtained with improved toughness and ductile properties, suitable for industrial use in the packaging sector and for medical applications too.Considering the intrinsic fragility of PHBH, the addition of different PCL wt %, allowed its toughness, ductility, and, above all, impact resistance to be improved. Blends with a 10 PCL wt % offered a percentage increase in the impact resistance of about 56%. The impact resistance was even improved up to double the initial value of neat PHBH by adding 40 PCL wt %. This effect of increasing the ductility of PHBH by increasing the PCL content in the PHBH/PCL blends had an opposite effect on mechanical resistant properties such as modulus and strength . In contrast, the elongation at break was remarkably improved, from 13.9% for neat PHBH up to 461% for the PHBH/PCL blend with 40 PCL wt %.Tg to be obtained, with two clear and unchanged values located at −47 °C (Tg of PCL) and at 9 °C (Tg of PHBH). These characteristic Tg values remained unchanged in the PHBH/PCL blends, thus suggesting lack of miscibility. Regarding the morphology of the PHBH/PCL blends, they did not show the typical drop-like PCL phase embedded in a PHBH matrix. PCL appeared in the form of small flakes which could exert a positive effect on ductile properties.On the other hand, thermal analysis suggested high immiscibility between PHBH and PCL, since the main thermal parameters of neat PHBH and PCL remained unchanged in blends, which is characteristic of very poor or lack of miscibility. Only a slight increase in the thermal stability of neat PHBH was obtained by adding different PCL wt %, since PCL is much more thermally stable than most biopolyesters. The dynamic mechanical thermal analysis (DMTA) allowed accurate values of


To evaluate the protective effect of melatonin on ovarian ischemia reperfusion injury in a rat model. Forty-eight rats were separated equally into 6 groups. Group 1: sham; Group 2: surgical control with 3-h bilateral ovarian torsion and detorsion; Group 3: intraperitoneal 5% ethanol (1 mL) just after detorsion (as melatonin was dissolved in ethanol); Group 4: 10 mg/kg intraperitoneal melatonin 30 min before 3-h torsion; Group 5:10 mg/kg intraperitoneal melatonin just after detorsion; Group 6:10 mg/kg intraperitoneal melatonin 30 min before torsion and just after detorsion. Both ovaries and blood samples were obtained 7 days after detorsion for histopathological and biochemical analysis.In Group 1, serum levels of total oxidant status (TOS) were significantly lower than in Group2 (P = 0.0023), while tissue TOS levels were lower than in Group 3 (P = 0.0030). Similarly, serum and tissue levels of peroxynitrite in Group 6were significantly lower than those ofGroup 2 . Moreover, serum oxidative stress index (OSI) (arbitrary unit) levels were significantly increased in Group 2 when compared to groups 1 and 6  and in Group 3 with respect to groups 1, 4, 5, and 6 . Furthermore, there was a significant decrease in histopathological scores including follicular degeneration, vascular congestion, hemorrhage, and inflammation in the melatonin and sham groups in comparison with control groups. Additionally, primordial follicle count was significantly higher in Group 6 than in Group 2 (P = 0.0002).Melatonin attenuates ischemia reperfusion damage in a rat torsion/detorsion model by improving histopathological and biochemical findings including OSI and peroxynitrite. Twisting of the ovary along with the fallopian tube is referred to as adnexal torsion; it may affect females of all ages, particularly women of reproductive age from 20 to 40 years of age [1]. The most common symptom of ovarian torsion is acute onset of lower abdominal pain, followed by nausea and vomiting. It presents clinically as a gynecologic surgical emergency and often compromises ovarian lymphatic and blood supply. Therefore, urgent diagnosis and treatment is crucial to preserve ovarian function and to prevent other related morbidities in patients with ovarian torsion. The most common method of treatment is laparoscopy, and the main objective of the intraoperative evaluation is to confirm the diagnosis of torsion and evaluate the viability of the ovarian tissue. Most twisted ovaries are considered potentially viable and can be preserved by detorsion unless there is an ovarian mass with suspicionof malignancy or the patient is postmenopausal, in which casesalpingo-oophorectomy is performed [2]. The blood supply to the twisted ovary is sufficient for survival of follicles despite its necrotic black bluish appearance [3].Untwisting of the twisted ovary reestablishes the blood supply; however, the return of blood flow can also result in further ovarian damage and complications, termed ischemia reperfusion injury [4]. Recently, many pharmacological agents have been found to alleviate the effects of ischemia reperfusion injury in animal models, due to their antioxidant and antiinflammatory effects [5–8]. Melatonin (5 methoxy-N-acetyltryptamine), discovered and isolated from bovine pineal gland by Aaron Lerner in 1958,is produced in many organs, mainly by the pineal gland . The circadian production of melatonin explains its regulatory action on circadian rhythms such as body temperature or sleep-wake cycles and neuroendocrine rhythms . Moreover, it has antioxidant and antiinflammatory properties and critical effects on many diseases, including asthma [13], diabetic retinopathy , infection [16], and endometriosis [17]. However, there have been only 3 studies in the literature showing preventive effects of melatonin on ischemia reperfusion injury of the rat ovarian torsion/detorsion model [18–20]. In addition, it has also been investigated in cat ovarian [21] and rat uterine [22] torsion/detorsion models.The objective of this study was to investigate the effectiveness of melatonin in preventing ovarian ischemia reperfusion injury in a rat model through biochemical and histopathological analysis.A total of 48 female adult Wistar albino rats weighing 150–250 g and aged 10–15 weeks were included in this study, which was performed at the Hacettepe University Department of Physiology Animal Research Laboratory. All experimental procedures were carried out once the study protocol was approved by the Hacettepe University Ethical Committee on Animal Research , with a commentary advising that both the sham and surgical control groups  should be the used in another similar study of ours (unpublished) performed at the same time period to decrease the number of rats used. The animals were fed ad libitum and kept in a temperature and humidity-controlled environment (22 ± 2 °C) with a cycle of 12 h of darkness followed by 12 h of light.Animals were allocated randomly into 6 groups. Group 1 : the abdominal wall was kept open for 3 h and then closed. Ovarian torsion was not performed. Rats were followed-up for 1 week after the first operation without any medication. Group 2 : bilateral ovarian torsion was performed; 3 h after ischemia, detorsion was applied. No treatment agent was given to the rats during the follow-up period of 1 week. Group 3 : bilateral ovarian torsion was performed; 3 h after ischemia, detorsion was applied. A single dose of 1-mL 5% ethanol solution was given intraperitoneally (i.p.) to all rats just after detorsion and administered daily for 1week .Group 4: 30 min before the induction of ovarian torsion, rats were given a single dose of 10 mg/kg i.p.melatonin . No maintenance treatment was applied after detorsion or during the follow-up period of 1 week. Melatonin in dry powder form was dissolved in 99% ethanol and then diluted in saline just before injection with final ethanol and melatonin concentrations of 5%and 10 mg, respectively, per mL,as in our previous studies [17]. Group 5 : a dosage of 10 mg/kg i.p. melatonin was given to each rat 30 min before the induction of ovarian torsion, and repeated just after detorsion preceding 3 h of ischemia. A 10 mg/kg i.p. dose of melatonin was administered daily for 1 week. Group 6 : a single dose of 10 mg/kg i.p. melatonin was administered to each rat just after the ovarian detorsion preceding 3 h of ischemia, and repeated daily during the 1-week period, postoperatively. Rats were anesthetized withan i.p. injection of a combination of ketamine hydrochloride  and xylazine hydrochloride .Once the abdomen was shaved and prepared with a povidone-iodine scrub, a laparotomy was performed by making a 2-cm longitudinal incision in the midline area of the lower abdomen, and the uterine horns and adnexa were viewed. Ischemia was induced for 3 h using a torsion model conducted by applying and rotating atraumatic vascular clips 360° clockwise to the vascular pedicle 1 cm above and below each ovary bilaterally. The abdominal wall including muscle aponeurotic plane and skin was sutured with 3-0 Vicryl . Bilateral oophorectomy was carried out 1week after the first surgery in all groups for histological scoring (right ovaries) and biochemical evaluation (left ovaries). A 1-mL blood sample was obtained from the vena cava of each rat for the measurement of biochemical markers. After the completion of the overdose of anesthetic drugs, all rats were sacrificed.Ovarian tissues were fixed in 10% formalin for 48 h. After fixation, the tissues were prepared using standard procedures and embedded in paraffin. Serial 5-μm sections were cut from ovary blocks with a microtome and stained with hematoxylin–eosin. For each specimen, all sections were analyzed using a light microscope with 10× magnification . Ovarian damage, including follicular cell degeneration, vascular congestion, hemorrhage, and inflammation (neutrophil infiltration), was scored histologically using a graduated scale .Total scores were calculated according to these parameters. Evaluation was performed by a pathologist who was blind to the study groups. μ mol H2O2 equivalent/gr protein)/TAS μ mol Trolox equivalent/gr protein × 10]. The peroxynitrite assay and protein content were determined, as previously described . The results were expressed as micromole/L for serum and micromole/g for wet tissue, respectively. Biochemical measurements were carried out using a spectrophotometer .Total antioxidant status (TAS) and total oxidant status (TOS) levels were measured using a colorimetric method applied by Erel [23]. The results are expressed in millimoles Trolox equivalent/L  for TAS, and micromolar hydrogen peroxide (H2O2) equivalent per liter of TOS  [24].The ratio of TOS to TAS was accepted as oxidative stress index (OSI). For calculation, the miilimole TAS unit was first converted to a micromole unit. OSI value was calculated according to the following formula: OSI (arbitrary unit) = . Thus, prevention of ovarian damage caused by ischemia reperfusion injury is important in the area of gynecology.Numerous therapeutic agents showing antioxidant actions have been studied for protective effects against ovarian ischemia reperfusion injury rat models [29–32]. Melatonin, an endogenous indolamine related to circadian rhythms, has proven itself as an essential agent against toxic free radicals and provides a strong defense against ischemia reperfusion injury . It was found in our previous study that melatonin caused regression of endometriotic implants in rats by improving antioxidation actions on superoxide dismutase and malondialdehyde [17].TAS and TOS reﬂect the total effects of all antioxidants and oxidants in the plasma, body ﬂuids, or tissues, respectively [23]. OSI is calculated with the formula of TOS/TAS and is regarded as a more appropriate index of oxidative stress on the plasma or tissue. Several antioxidant free radical scavengers have been shown to prevent ovarian ischemia reperfusion injury in rats by improving TAS, TOS, and OSI . Peroxynitrite (ONOO–) is the major component of nitroxidative stress. When reactive nitrogen species are overproduced, nitric oxide (NO•) reacts quickly with the superoxide radical (O2•–) to form peroxynitrite (ONOO–), which is itself cytotoxic, and then easily decomposes into toxic hydroxyl radical (OH •) and nitrogen dioxide. As peroxynitrite exerts its cytotoxic action thru mitophagy, inhibiting peroxynitrite-mediated mitophagy activation improves ischemia reperfusion injury . Moreover, melatonin as an antioxidant improves TOS, TAS, OSI, and peroxynitrite, as demonstrated in previous studies [39–41].To the best of our knowledge, protection of the rat ovary from ischemia reperfusion injury using melatonin has been investigated in only 3studies up to date. In thefirst study, histopathological scores and malondialdehyde were significantly improved by melatonin in a rat torsion/detorsion model [18]. In the second study, melatonin (10 mg/kg i.p. for 3 h) was found to protect the ovaries against oxidative damage associated with reperfusion following an ischemic insult in rats, based on the histopathological and biochemical ﬁndings including malondialdehyde, glutathione, and xanthine oxidase [19]. Similarly, malondialdehyde, myeloperoxidase, glutathione, glutathione peroxidase, glutathione reductase, glutathione s-transferase, and superoxide dismutase were found to be significantly improved by 2.2 mg/kg i.p. melatonin in the third study [20]. Moreover, it has been reported that melatonin decreased cat ovarian [21] and rat uterine [22] tissue injury after detorsion.In our current study, rats were divided into 6 groups: the sham group; the torsion/detorsion group; 3 melatonin (10 mg/kg i.p.) groups . The rationale behind scheming our melatonin groups as the prophylaxis, maintenance, and prophylaxis plus maintenance groups in our study was to mimic real clinical scenarios in which a patient attends with the suspicion of adnexal torsion, (i) the physician can give the therapeutic agent before detorsion for prophylactic treatment, (ii) the physician can give the therapeutic agent only after detorsion as a maintenance treatment, or (iii) the physician can give the therapeutic agent not only before detorsion for prophylactic treatment, but also daily after detorsion for a while for maintenance treatment (prophylaxis plus maintenance treatment). However, there was no significant difference between these treatment groups.The results of this study have shown that melatonin reduced ischemia reperfusion damage in the rat ovary by histopathological and biochemical parameters as shown for the third time in the literature. However, our study is the first in the literature to show that melatonin prevented ischemia reperfusion in a rat ovarian torsion/detorsion model by improving oxidative stress in both tissue and serum levels of peroxynitrite and serum levels of OSI. Moreover, the melatonin group had a significantly higher primordial follicle count than the surgical control group.This study has several limitations. An experimental rat model of ovarian torsion/detorsion cannot really exemplify the classical pathophysiology in clinical practice in humans. In addition, there is no consensus on the optimal dose and optimal duration of treatment with melatonin. Moreover, a 7-day follow-up period corresponds to a length of approximately 2 menstrual cycles, as the estrous cycle of the rats takes about 4–5 days. However, it would be improved if the rats were followed up with for a longer period of time. Furthermore, it would be better to measure primordial follicle count to see the effects of ischemia reperfusion injury. Rotation of the pedicle may not be needed to induce ischemia reperfusion injury after vascular clip usage, as it might have caused vascular injury in this experimental model. Additionally, intraperitoneal alcohol is known as a sclerosing agent, but it was demonstrated that even intraperitoneal 99.9% ethanol had no more severe side effects [42]. Moreover, the ethanol used in this study was only 5% in concentration. Thus, further prospective animal and even human studies are needed to determine the value of melatonin for protecting ovaries from ischemia reperfusion injury after detorsion.All in all, our results show that melatonin exerts protective effects against ischemia reperfusion injury in a rat torsion/detorsion model by improving histopathological and biochemical findings, including primordial follicle count, tissue and serum levels of peroxynitrite, and serum levels of oxidative stress index.Conflict of interestThe authors report no conflict of interest with any funding agency or commercial sectors.


The experiment lasted 30 days, with 20 days adaptation and 10 days stool and blood collection. Results were analyzed for normality and means were separated by ANOVA and adjusted by the Tukey test at the significance level of 5.0%. Prebiotic supplementation had no effect on apparent digestibility coefficients (ADC), total stool production and fecal scores (p > 0.05). Prebiotics evaluated also did not alter fecal pH, nor the concentrations of ammonia, lactic acid, short chain fatty acids (SCFA) and most fecal branched chain fatty acids (BCFA) (p > 0.05). The addition of GOS decreased the concentration of iso-valeric acid (p = 0.0423). Regarding immunological variables, concentrations of fecal IgA were not influenced by the treatments. Treatments GOS and B2 increased the total number of polymorphonuclear cells, as well as the oxidative burst in relation to treatments B1 and CO (p < 0.0001). Treatment B2 improved the rate of S. aureus phagocytosis in relation to CO (p = 0.0111), and both the GOS and B2 treatments had a better index for E. coli phagocytosis than the CO treatment (p = 0.0067). In conclusion, there was indication that both prebiotics GOS and B2 at 1.0% inclusion improved the immunity of healthy dogs.This study aimed to evaluate the effects of two prebiotics in different concentrations on nutrient digestibility, fermentative products and immunological variables in adult dogs. Twenty-four adult dogs were randomly divided into six blocks according to their metabolic body weights (BW Prebiotics represent some of the most common functional ingredients used in pet foods. These can be defined as substrates used selectively by host microorganisms that confer gut health benefits . PrebiotBesides promoting direct positive effects on intestinal health, prebiotics can indirectly improve the animal's immune system by stimulating the growth of lactic acid-producing bacteria. These bacteria produce substances with immunostimulatory properties, which interact with the immune system and stimulate cytokine production, mononuclear cell proliferation, macrophage phagocytosis and induction of synthesis of larger amounts of immunoglobulins .Bifidobacterium proliferation in the colon, which suppress the activity of putrefying bacteria by antagonistic effect and reduce the formation of toxic metabolites [Galactooligosaccharides (GOS) are prebiotics synthesized from lactose transgalactosylation. Recent studies attribute to these oligosaccharides a number of potential health benefits . Galactoabolites , 8. OtheIn order to optimize the isolated effects of certain prebiotics it is possible to use these as blends. When incorporated into the animal's diet, blends can modulate the microbiota, improve the animal’s intestinal health and immunity, and confer additional benefits of each different prebiotic . Thus, tThis study was in agreement with the ethical principles in animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and the Ethical Principles in Animal Research established by the Ethic Committee on Animal Use of the School of Veterinary Medicine and Animal Science at the University of São Paulo (CEUA/FMVZ). The study was approved by the CEUA under the protocol number 5359160216.ad libitum. During the collection period, dogs remained in the same kennels and were individually released for one hour in exercise areas, accompanied by a student. When dogs defecated, feces were immediately harvested.The experiment was conducted at the Premier Pet Nutrition Development Center . Twenty-four mixed-breed healthy male and female dogs were selected, with a mean age of 4.0 ± 2.0 years and body condition score between 4 and 5 . Dogs we0.75). Within each block, dogs were randomized to four treatments: CO , GOS , B1 (control treatment with 0.5% Yes-Golf® prebiotic blend addition), B2  and body condition score (BCS).The feeding study lasted for a total of 30 days, with the first 20 days of adaptation to foods. From day 21 to 25, total fecal samples were collected for measurement of apparent digestibility. From day 26 to 29, fresh feces were collected into 2mL vials and immediately frozen at -80ºC for later determination of fermentation products. On the last day (day 30), 5mL blood was collected from jugular venipuncture according to  recommenDietary ADCs were determined by the total fecal collection method according to . In summThe dry matter (DM), crude protein (CP), acid-hydrolyzed fat (AHF), ash and crude fiber (CF) contents were determined in both feces and food  accordinAll stools collected during the digestibility study were scored for consistency on a 5-point scale (0 to 5), with formed and firm  stools being between 3 and 4 .Fecal pH was determined by homogenizing one gram of fresh feces with 9mL distilled water, and introducing the electrode into 2 points of the solution  using a Fecal ammonia nitrogen and short chain fatty acids (SCFA) were determined on fresh homogenized fecal samples, collected within 15 minutes after defecation. Immediately upon collection, three grams of duplicated fecal samples for each parameter measured  were mixed with 9mL 16% formic acid. The mixture was kept in a refrigerator for seven days and stirred daily. Subsequently, the mixture was centrifuged at 5,000 rpm for 15 minutes at 15°C three times, discarding the pellet. The supernatants were extracted, identified, and stored at -15°C. For fecal ammonia nitrogen quantification, the extracts were thawed at room temperature, 2mL aliquots were diluted in 13mL distilled water, and then processed in a nitrogen distiller according to [For SCFA determination, the last supernatant was transferred to an Eppendorf tube  before freezing and storage. At the end of all periods, all samples were thawed and centrifuged at 14,000 rpm  for 15 min. Fecal SCFA concentrations were analyzed by gas chromatography , according to  and adapLactic acid was measured according to the methodology described by . BrieflyImmunological assays included blood leukocyte, total polymorphonuclear cells, intracellular production of reactive oxygen species (ROS) and blood leukocyte phagocytosis. These analyzes were performed by flow cytometry , according to the methodology described by . On the Phagocytosis tests were conducted by adding 2μL of Staphylococcus aureus or Escherichia coli already labeled with fluorescent reagent Alexa Fluor® conjugate to the polypropylene tubes. These were incubated for 60 minutes at 37ºC, and then the reactions were stopped by the addition of 2,000μL cold ethylenediaminetetraacetic acid (EDTA) solution (3mM). The tubes were centrifuged at 250 x g for eight minutes and the supernatants discarded. Then, samples were homogenized, and the red cells were hypotonically lysed with saline solution . After this procedure, samples were centrifuged twice. Finally, the samples were read on a FACS Calibur TM flow cytometer  connected to an Apple Macintosh computer  with the CELLQUEST® - Becton Dickinson Immunocytometry System TM program . A total of 10,000 cells were acquired from each tube and the data obtained from the readings were analyzed on FlowJo Treestar—vX.0.7 version for Windows software . These analyzes were performed at the Immunodiagnostic Laboratory of the Department of Veterinary Clinic of FMVZ/USP .In addition to the immunoassays performed on blood, fecal IgA was also determined. For this, 3g of fresh fecal sample (within 30 minutes of defecation) were collected and frozen at -15ºC. Samples were thawed on the day of the analysis and IgA was extracted with saline solution according to . In summThe results were analyzed using the Statistical Analysis System  computer program . The norIn which:All animals had adequate food intake and no food rejection or diarrhea were reported. There were no outliers, so all observations were kept for statistical analysis. During the experiment, dog weights and body condition scores were monitored and maintained. There was no difference between the treatments in the ADC variables of DM, OM, CP, AHF and NFE (P> 0.05), as well as in wet fecal production and fecal scores  in the concentration of most fermentation products measured in the study  and fecal IgA were not different among treatments , the percentage of cells that phagocytized at least one bacterium was higher in dogs fed the B2 diet than those fed the negative control ; however, group B2 did not differ from groups GOS and B1 (P > 0.05). The GOS and B2 groups presented higher means than CO in the phagocytosis test with gram negative bacteria (P = 0.0067), and B1 was similar to the extremes. Finally, the oxidative burst, for both gram-positive and gram-negative stimuli GOS and B2 presented higher fluorescence intensity. This translates to a higher amount of ROS produced in dogs fed the GOS and B2 treatments due to more intensive phagocytoses in comparison to those fed the B1 and CO diets. The oxidative burst is measured by flow cytometry through the immunofluorescence intensity emitted by the contact of ROS with the fluorescence reagent of pre-treated bacteria (E. coli or S. aureus). When the bacteria is phagocyted, the cell generates ROS and the color intensity changes. The ROS produced during phagocytosis has antimicrobial activity due to microbial DNA and protein damage [The percentage of polymorphonuclear cells in relation to total leukocytes was higher in animals that consumed the GOS and B2 diets (P < 0.0001). Regarding the phagocytosis test with gram positive bacteria  had an increase in the relative number of polymorphonuclear cells, as well as an improvement in the phagocytosis index and an increase in ROS production for both gram-positive and gram-negative bacteria stimuli. Few experiments have assessed the effect of prebiotics on the immune response of healthy dogs. The pioneering study on this field found a higher concentration of lymphocytes in dogs fed the prebiotic treatments  compared to the control . In the Cyprinus carpio) increased innate immunity, total Ig and significant level of lysozyme [It has been reported that GOS may improve immune responses through SCFA production , 35. Gallysozyme . In addilysozyme –39.In addition to GOS, the blend used in this study contained MOS, FOS and β -glucan which have also shown evidence of immunological improvement. Some specific prebiotic compounds (mannan and glucans) perform antibacterial, antimutagenic, antitumor and antioxidant functions . BesidesThe FOS present in treatment B2 may also have influenced the immune system of dogs. It was reported that this prebiotic interacts with Toll-like receptor-2 (TLR2), a membrane surface receptor expressed in macrophages, polymorphonuclear cells (polymorphonuclear leukocytes or granulocytes) and dendritic cells, which results in the activation of immune cells through pathways of signal transduction in humans .Finally, β-glucan can act as an immunostimulant and activate immune cells by binding to their specific lectin-1 receptor. This is a type C lectin receptor expressed on the surface of macrophages , 46. TheAccording to the results obtained in this experiment, dogs fed the treatments GOS and B2 had an increase in the concentration of polymorphonuclear cells, the number of cells that performed at least one phagocytosis, and in the oxidative burst measured by flow cytometry. Besides that, treatments containing prebiotics did not affect nutrient digestibility and fecal characteristics. In conclusion, the prebiotic blend and GOS at 1% led to the greatest improvements in immunity, suggesting that these prebiotics should be employed at a concentration greater than 1% to promote health.


Wearable devices equipped with inertial sensors enable objective gait assessment for persons with multiple sclerosis (MS), with potential use in ambulatory care or home and community-based assessments. However, gait data collected in non-controlled settings are often fragmented and may not provide enough information for reliable measures. This paper evaluates a novel approach to (1) determine the effects of the length of the walking task on the reliability of calculated measures and (2) identify digital biomarkers for gait assessments from fragmented data. Thirty-seven participants (37) diagnosed with relapsing-remitting MS (EDSS range 0 to 4.5) executed two trials, walking 20 m each, with inertial sensors attached to their right and left shanks. Gait events were identified from the medio-lateral angular velocity, and short bouts of gait data were extracted from each trial, with lengths varying from 3 to 9 gait cycles. Intraclass correlation coefficients (ICCs) evaluate the degree of agreement between the two trials of each participant, according to the number of gait cycles included in the analysis. Results show that short bouts of gait data, including at least six gait cycles of bilateral data, can provide reliable gait measurements for persons with MS, opening new perspectives for gait assessment using fragmented data . Stride time variability and asymmetry, as well as stride velocity variability and asymmetry, should be further explored as digital biomarkers to support the monitoring of symptoms of persons with neurological diseases. Gait impairment is highly prevalent in multiple sclerosis (MS), as the decline in neural control affects motor functions, and consequently gait, balance and mobility ,2. ObjecData collected from wearable devices, equipped with inertial sensors, have been demonstrated to be effective in objective gait assessments, offering a portable and cost-effective solution compared to large or fixed installations ,5,8. HowOne approach to address this challenge is to select short bouts of gait data, representing predefined number of gait cycles, from the full length of a walking task. The analysis of the intrasession reliability can show the effects of length of gait data on the reliability of gait parameters, to identify the spatiotemporal gait parameters that can be used to obtain reliable measures according to the number of gait cycles included in the analysis.The selected approach focuses on the study of the reliability of spatiotemporal gait parameters from a limited number of gait cycles, which could, therefore, be applied to fragmented data, enabling gait assessments with wearable devices. The main goal of the present study is to define an optimal length of gait data and identify the spatiotemporal gait parameters that can be used to define digital biomarkers for persons with MS.Participants were recruited from the neurology outpatient department at St. Vincent’s University hospital, Dublin, Ireland. The inclusion criteria were participants diagnosed with clinically definite relapsing-remitting MS, able to execute two 20 m walking trials safely without a mobility aid. All participants provided informed consent, and ethical approval was obtained from St. Vincent’s hospital research ethics committee.The present study includes data from 37 participants with MS—mean age 45.1 ± 9.9, height 168.6 ± 9.9 cm, weight 75.5 ± 16.8 kg, 23 females (62%); mean time since diagnosis was 7.4 ± 7.7 years. Each participant received a comprehensive neurological and physical examination including Expanded Disability Status Score (EDSS), which quantifies disability in people with MS ranging from 0  to 10 (Death due to MS). EDSS steps from 1.0 to 4.5 refer to people with MS who are fully ambulatory . At the time of the assessment, 13 participants had EDSS score 0, 13 had EDSS scores 1 or 1.5, and 11 had EDSS scores 2 or above. Mean time to complete a TUG test was 7.8 ± 1.7 s, while mean stride velocity was 122.2 ± 15 cm/s and mean stride length 136.6 ± 16.9 cm. A table of clinical information for the participants is available as To include measures of gait symmetry, the study protocol was designed to include a collection of bilateral gait. Two inertial sensors were attached to the participant’s right and left shanks, at the midpoint of the anterior shank using dedicated Velcro straps. Sensors sampled at 102.4 Hz and contained a triaxial accelerometer and a triaxial gyroscope. Data were streamed in real time via Bluetooth using dedicated software  and stored for offline analysis.Participants were instructed to walk at their preferred self-selected pace, starting with their dominant foot (right or left). Each participant completed two trials of the 20 m walking task, in the same day, with a short break between trials.Previously published algorithms were selected for the procedures for calibration, data treatment and artefact rejection ,13,14.The gait event detection creates a sequence of Initial Contact (IC) and Terminal Contact (TC) points corresponding to the movements of each leg. ICs and TCs represent heel-strikes and toe-offs, respectively, and define the phases of stance (IC to TC) and swing (TC to IC) for each leg.The data extraction process ignored the first gait cycle  and included data from the 3rd IC. Following the gait event detection, ICs and TCs were used to calculate spatiotemporal gait parameters, as described: mean swing time , mean stance time , stride time . Mean step time represents average of times between IC of one foot to IC of the opposite foot, in seconds. Mean single support is the proportion of gait cycle spent on either foot and mean double support is the proportion of gait cycles spent on both feet, averaged across multiple gait cycles. Spatial information was estimated from stride length. Mean stride length (m) and mean stride velocity (cm/s) were calculated and averaged across both legs.For gait variability, the coefficient of variation (CV) was calculated as the standard deviation (SD) divided by mean values within participants across multiple gait cycles, as a percentage. Gait variability was calculated for stride time, stance time, swing time, step time, single support, and double support.For gait asymmetry, the Gait Symmetry Index (GSI) represents the difference between right and left divided by the average of right and left values, expressed as a percentage. Minus values indicate left leg asymmetry . Gait syA table with the definitions of the spatiotemporal gait parameters for the present study is available as The intrasession reliability was calculated using intraclass correlation coefficients ) ,17. The Based on the ICC estimate, the reliability of gait parameters was described as “poor” (less than 0.5), “moderate” (between 0.5 and 0.75), “good” (between 0.75 and 0.9) or “excellent” (0.9 and greater) .Participants executed different numbers of steps to complete the walking task. In order to calculate the ICCs for all the spatiotemporal gait parameters, and include all the trials, a minimum number of three and a maximum number of nine complete gait cycles  could be extracted from the full length of the walking tasks. ICCs were then calculated at predefined numbers of gait cycles: 3, 4, 5, 6, 7, 8 and 9.The mean time for all the participants to complete each walking task was 16.1 ± 3.2 s. Mean stride velocity (excluding time from recording start to gait initiation) was 146.2 ± 23.5 cm/s, and mean stride length was 135.6 ± 18.5 cm across all trials.Spatiotemporal gait parameters representing average values across gait cycles reached “excellent” reliability from three gait cycles , as shown in Stride length variability reached “good” variability from three gait cycles, while all the other parameters describing gait variability showed increased reliability when more gait cycles were included in the analysis. Some gait variability parameters, in particular stance time variability, swing time variability, step time variability and stride velocity variability, reached “good” reliability after six gait cycles and tended to continue towards an excellent reliability around nine gait cycles, with an exception of the variability of double support, as shown in Reliability of parameters describing stride length asymmetry and stride velocity asymmetry reached “good” reliability from four gait cycles, while stance time asymmetry and swing time asymmetry reached “good” reliability from seven gait cycles. Step time asymmetry showed “moderate” reliability even when nine gait cycles were included in the analysis. In the present analysis, the calculation of stride time asymmetry showed “poor” reliability. The reliability of gait asymmetry parameters is presented in In the present study, we analysed the intrasession reliability of spatiotemporal gait parameters  for assessing gait of participants with MS according to a predefined number of gait cycles, representing short bouts of gait data extracted from the full walking task. The main goal of this analysis is to evaluate a novel approach for gait analysis from data collected with wearable devices, supporting gait assessments in ambulatory care and less controlled settings, such as patients’ homes or community ambulation, where gait data are often fragmented .The analysis of the reliability of spatiotemporal gait parameters followed the extraction of predefined numbers of consecutive gait cycles from the full walking task, representing short bouts of gait data. This approach has been presented in the literature in order to assess the reliability of measures collected from fragmented data . AnotherAs different test conditions can affect the calculated measures ,22, therIn the literature, many authors investigated the reliability of spatiotemporal gait parameters, in particular for measures of gait variability, recommending calculated measures should be carefully reported together with the length of the walking tasks and the test conditions ,24,25. DThe analysis showed that spatiotemporal gait parameters calculated as the average across gait cycles, and representing bilateral gait collected from inertial sensor data, can reach “excellent” reliability from as few as three gait cycles. This result is in line with the literature, describing “good” test–retest reliability for mean stride length, mean stride velocity, mean stance time, mean swing time and mean double support for participants with MS .The present analysis of the reliability of parameters describing gait variability presents stride time variability, stride length variability and stride velocity variability reaching “good” and “excellent” reliability with fewer strides than previous results reported in the literature ,25. InclOne goal of the present study is to determine which spatiotemporal gait parameters can provide reliable gait measurements for short bouts of gait data collected from wearable devices.As stressed by Frechette et al. (2019), in order to provide relevant clinical data for the assessment of MS related symptoms, it is crucial to focus on gait quality over quantity . Our curA current challenge for wearable technologies is the form factor, enabling them to be worn for long periods of time. Technological limitations such as battery life, weight, shape and material, mean that most wearable devices would only enable data collection for short periods of time, whether used in controlled or in non-controlled settings. One exception is devices strapped to the user’s wrist, which users agree to wear for extended periods of use. However, the wrist worn devices have not been proven to provide reliable measures for gait quality assessment, nor have been studied for patients with mobility impairment. The present study recorded bilateral data from participants’ right and left shanks, which provides complimentary information to evaluate gait asymmetry.While stride time asymmetry and step time asymmetry have “poor” reliability, results show that gait asymmetry estimated from stance time, swing time, stride length and stride velocity should be further explored, since they provide reliable measures for short bouts of gait data and have the potential to diagnose and monitor symptoms of persons with MS .Our study extends the findings of Strom et al. (2018) on the analysis of the effects of the length of walking bouts on the measures of step-by-step characteristics . The evaAccording to Storm et al., 2018, daily living gait is characterised by higher pace compared to studies observed in controlled settings , regardless of the duration of the walking bouts. Concerning the variability of spatiotemporal gait parameters, there was no significant difference between controlled and non-controlled settings, for continuous or intermittent walking. This analysis reinforces our promising result, showing that measures of gait variability and gait asymmetry (in particular of stride length and stride velocity), should be further investigated for their utility in neurological assessment of persons with MS and other neurological conditions.The present study shows that short bouts of gait data, representing bilateral gait data collected using inertial sensors, can provide reliable measures for objective gait assessment of persons with MS.Gait parameters representing average values across gait cycles reached “excellent” intrasession reliability from three gait cycles, while parameters describing gait variability and asymmetry tended to reach higher ICCs when more gait cycles were included in the analysis.From six gait cycles, stride length variability and asymmetry, as well as stride velocity variability and asymmetry, show “good” reliability and should be further explored with regard to their potential contribution to the early diagnosis and monitoring symptoms of persons with MS. Stride time asymmetry and step time asymmetry do not seem to provide reliable measures and should be reported carefully.The main contribution of the present study is to demonstrate that short bouts of gait data, including at least six gait cycles of bilateral data, can provide reliable gait measurements for persons with MS, opening new perspectives for wearable devices and digital biomarkers for gait assessment in non-controlled environments, to support the monitoring of symptoms of persons with neurological diseases.