Feed efficiency is an important economic trait and reduces the production costs per unit of animal product. Up to now, few studies have conducted transcriptome profiling of liver tissue in feed efficiency-divergent chickens (Ross vs native breeds). Also, molecular mechanisms contributing to differences in feed efficiency are not fully understood, especially in terms of long non-coding RNAs (lncRNAs). Hence, transcriptome profiles of liver tissue in commercial and native chicken breeds were analyzed. RNA-Seq data along with bioinformatics approaches were applied and a series of lncRNAs and target genes were identified. Furthermore, protein–protein interaction network construction, co-expression analysis, co-localization analysis of QTLs and functional enrichment analysis were used to functionally annotate the identified lncRNAs. In total, 2,290 lncRNAs were found , of which 53 (including 39 known and 14 novel), were identified as differentially expressed genes between two breeds. The expression profile of lncRNAs was validated by RT-qPCR. The identified novel lncRNAs showed a number of characteristics similar to those of known lncRNAs. Target prediction analysis showed that these lncRNAs have the potential to act in cis or trans mode. Functional enrichment analysis of the predicted target genes revealed that they might affect the differences in feed efficiency of chicken by modulating genes associated with lipid metabolism, carbohydrate metabolism, growth, energy homeostasis and glucose metabolism. Some gene members of significant modules in the constructed co-expression networks were reported as important genes related to feed efficiency. Co-localization analysis of QTLs related to feed efficiency and the identified lncRNAs suggested several candidates to be involved in residual feed intake. The findings of this study provided valuable resources to further clarify the genetic basis of regulation of feed efficiency in chicken from the perspective of lncRNAs. In response to the global demand for the chicken meat, genetic selection enabled us to improve many traits related to the functions of chickens and led to faster growth rates and higher feed efficiency in different commercial races such as Ross3. On the other hand, given the importance of native chicken breeds in rural economies in most of the developing and underdeveloped countries in having greater resistance to disease and the ability to tolerate harsh environmental conditions, their genetic improvement is reasonable in order to increase their efficiency. In this regard, Esfahan chicken is one of the native and dual-purpose breeds (to produce meat and eggs) in Iran4. Previous studies have demonstrated that feed efficiency is affected by many factors including food intake, genetic diversity in energy requirements for maintenance, the digestion, metabolism of nutrients, diversity in body composition and body temperature regulation5. Changes in the amount of energy required to digest food, the size of the digestive organs, and the amount of energy consumed within the tissues can all contribute to feeding intake variation6. Herein, birds with low-level RFI (Ross) means to consume less feed than predicted and are identified as efficient birds7. Furthermore, compared to the low efficient chickens, high efficient one may synthesize ATP more efficiently and control reactive oxygen species (Ross) production more strictly by enhancing the mitochondrial function in skeletal muscle8. According to the importance of Esfahan chickens compared to commercial breeds in having more resistance to diseases, efforts to improve chickens with efficient performance and a better feed efficiency would be beneficial with emphasis on genetic improvement. In this context, understanding the genetic mechanisms underlying the differences between native and commercial chicken breeds can be a promising strategy for a quick genetic improvement of the Esfahan breed.Along with the increase in the world's population and the growing need for food, in particular, protein substances, meeting human nutritional needs is primarily an issue for human societies. Chicken meat is a high-quality protein source for humans due to the high levels of protein, low cholesterol and low calories. The poultry industry plays a vitally important role in converting grains into meat and eggs, which are essential sources of food for humans9. One type of important ncRNAs that have been taken into consideration is Long noncoding RNA (lncRNA). LncRNAs are mostly defined as RNAs greater than 200 nucleotides in length, without any coding potential10. Most of the lncRNAs are processed like mRNA to be 5′ capped and 3′ poly adenylated as well as to be alternatively spliced12. In spite of lacking protein-coding potential, recent studies have shown the presence of short open reading frames (ORFs) that encode small peptides with vital biological functions. It has been reported that lncRNAs are less evolutionary conserved and tend to be expressed lower than that of protein-coding genes, as most of them are tissue specific. The most important functions of lncRNAs are regulating gene expression (by cis or trans-acting) through epigenetic, transcriptional, post-transcriptional, or post-translational mechanisms14.Gene expression profiling is a powerful approach in functional genomics to uncover the complexity of cells and tissues at the transcriptional level. Next generation sequencing (NGS) based technologies are revolutionizing the field and paved the way to profile the gene expression of many organisms. In this context, RNA sequencing (RNA-Seq) is the current gold-standard method for genome-wide gene expression studies, which takes advantage of NGS. Hence, transcriptome analysis can help us to identify the molecular mechanisms affecting different traits such as feed efficiency. On the other hand, it is well known that the majority of the genome is expressed to non-coding RNAs (ncRNAs), which play critical roles in various biological processes15. In a recent study, Muret et al., for the first time, reported a new lncRNA (lnc_DHCR24) as a key enzyme of cholesterol biosynthesis in chicken lipid metabolism16. Also, a study on six Beijing-you cocks, divergent in sperm motility led to the identification of 2,597 lncRNAs, including 1,267 intergenic lncRNAs (lincRNAs), 975 anti-sense lncRNAs and 355 intronic lncRNAs (ilncRNAs). Of these, 124 were differentially expressed and two lncRNAs including MSTRG.3652 and MSTRG.4081 and their target genes, showed their potential in sperm motility regulation17. A recent genome-wide study identified 9,393 tissue-specific lncRNAs (including 5288 novel genes) in eight tissues of chicken18. Moreover, 38 long intergenic non-coding RNA (lincRNAs) were reported in the duodenal transcriptome architecture of extreme RFI phenotypes in the six brown-egg dwarf hens19. In addition, many studies have suggested that lncRNAs play an important controlling role during lipid metabolism20, carbohydrate metabolism22, growth traits23, liver development24, ion transfer26 and digestive enzymes23 in chickens, which can affect feed efficiency in this way. In spite of abundant researches on feed efficiency of another farm animals28. limited researches have been conducted on the regulatory roles of lncRNAs in feed efficiency of chicken, especially in native breeds. Therefore, more studies that adopt genome-wide approaches are required to gain greater insight into these mechanisms. Additionally, there is a lack of information concerning the differences between native and commercial chicken breeds. In this study, RNA-Seq technology was applied to compare expressed lncRNAs in the liver tissue of two chicken breeds . The main goal was to gain a better understanding of biological and molecular mechanisms that are involved in the feed efficiency between native and commercial chicken breeds and establishing a foundation for future molecular studies. Moreover, using a stringent bioinformatic pipeline, novel lncRNAs were predicted, which can improve the annotation of the chicken genome.A growing number of reports have demonstrated regulatory functions of lncRNAs in different chicken tissues using RNA-Seq. For instance, Li et al., identified differentially expressed lncRNAs from chicken skeletal muscle and reported that these genes might play important roles in the muscle development29. Briefly, liver tissues of the chickens  were collected after the chickens were slaughtered. The chickens of both groups were raised in a birdhouse located in Safiabad Agricultural and Natural Resources Research and Education Center under the same environmental  and dietary conditions with ad libitum access to the diet and water, for 42 days. To better understand the difference in the growth and feeding characteristics feed intake DFI and BWG of each individual were monitored on a daily basis from 24 to 42 d of age. All tissue samples were snap frozen in liquid nitrogen and then transferred to a − 80 °C freezer until required for RNA isolation. Total RNA was extracted from the tissue samples using TRIzol reagent  based on the manufacture’s protocol. Three RNA samples from the same breed were pooled  to generate a total of four pooled RNA samples (two samples in each breed) and were sent to BGI company . Samples were applied for sequencing if the RNA Integrity Number (RIN) was > 7, based on an Agilent Bio Analyzer 2100 system. All the samples were subjected to a paired-end sequencing of 150 bp on the Illumina HiSeq 2000 platform . The raw RNA-Seq data were deposited and released in SRA database, with the BioProject accession number of PRJNA707148 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA707148).RNA-Seq raw data used in this study was obtained from our previous study. Detailed information about the samples and the experimental design has been described previouslyhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/)30. Raw reads were processed for initial trimming and filtering of the low-quality reads using the Trimmomatic tool (v 0.35)31. Then, STAR software (version 2.5.3.e) was applied to align the clean reads to the reference genome  using the default parameters32. StrnigTie tool (version 1.3.4.d) was used to assemble the aligned reads for each sample into transcripts33. Then, all the assembled transcripts of four samples were merged using the StrnigTie tool to generate a global and unified set of transcripts across the samples. This approach maximizes the overall quality of the final assembly. Differential expression analysis of the annotated genes was performed using Cuffdiff tool (v.2.2.1)34. To improve differential expression analysis for less abundant transcripts, the upper quartile normalization option was chosen. Finally, genes with a false discovery rate (FDR) ≤ 0.05 were considered as differentially expressed genes (DEGs) between the two breeds34. The reference genome for chicken and the annotation GFT file (release 91) were downloaded from the Ensemble database (https://www.ensembl.org/index.html)33.A computational approach using different filter criteria was applied to identify the lncRNAs Fig. . Prelimihttps://github.com/gpertea/gffcompare). Using this approach annotated lncRNAs were found. Then, only the unknown intergenic (marked with class code “u”) and intronic (marked with class code “i”) transcripts were subjected to further analyses. All unknown transcripts were blasted against NONECODE database (v5.0) (http://www.noncode.org) to identify the reported chicken lncRNAs in this database. It is worth noting that there are a large number of chicken lncRNAs in this database that are not included in the Ensemble chicken genome annotation. All the significant hits  were considered as known lncRNAs and were further classified to intergenic lncRNAs (lincRNAs) and intronic lncRNAs (ilncRNAs) based on their class codes. Different kinds of lncRNAs based on their genomic origins relative to nearby protein-coding genes can be grouped as sense or antisense transcript overlapping and as promoter associated, intronic or intergenic35. An ilncRNA possesses a transcribed region overlapped with the intron segments of protein-coding genes, and a lincRNA is located in the intergenic region lacking known protein-coding genes36. Also, lincRNAs have high relative abundance in the genome, for instance37. Kuo et al. in their study found that lincRNAs accounted for more than half of chicken lncRNAs. For the remaining transcripts, longer than 200 nt, the following stringent filtering criteria were performed to identify the potential novel lncRNAs37. First, the transcripts with one exon that were longer than 10,000 nt or those were located in the genomic regions containing simple sequence repeats (based on UCSC RepeatMasker file) were removed. Second, the transcripts with FPKM ≥ 1 expressed in at least two samples were kept. Third, all the remaining transcripts that were blasted  against one of the following databases were excluded from further analysis: UniprotKB (https://www.uniprot.org/help/uniprotkb) (by BLASTx), miRbase (https://www.mirbase.org/)  and Rfam (http://rfam.xfam.org/) (by BLASTn)38. Also, Hmmscan tool from the HMMER package (version 3.1b2) was applied to evaluate which of the remaining transcripts contains a known protein domain with any of the known protein family domains in the Pfam database39. The significant hits were then removed. Forth, to predict the coding potential of the remaining transcripts, five software including CPC2 (version 2 beta) (score > 0.5)40, CNCI (version 2) (score > 0)41, CPAT (version 1.2.4) (score > 0.36)42, PLEK (version 1.2) (score > 0)43 and FEElnc (version 12/07/2016) (default parameters)44 were used. Only the transcripts that were simultaneously considered to have coding potential by at least three of the mentioned tools were excluded from further analysis. Next, putative protein-coding transcripts were filtered out by considering open reading frame (ORF) length greater than 300 aa, which was predicted using TransDecoder tool (v2.1.0) (https://transdecoder.github.io/). In the last step, the intergenic transcripts that were located in a distance < 1 kb to a known protein-coding gene, were excluded. Finally, all the transcripts that successfully passed the filtering steps were considered as putative novel lncRNAs and classified into lincRNAs and ilncRNAs classes  by Gffcompare . The hits meeting the E-value threshold < 10–5 were considered significant.Genomic synteny analysis It is well known that establishing homology relationships among lncRNAs is challenging due to their low sequence conservation. Previous studies suggested that genomic position (synteny) of lncRNAs is more conserved than their cross-species sequence conservation. Therefore, lncRNAs conservation is geared toward the maintenance of synteny47. In other words, suppose neighboring upstream and downstream protein coding genes of a lincRNA in chicken are X and Y. If there be a lincRNA in other organisms that is located between the same X and Y protein coding genes, it can be considered as a conserved syntenic lincRNA. Here, to identify syntenic lncRNAs, order of surrounding genes flanking each putative novel lincRNA was compared for conserved synteny in the human and bovine genomes. All the genomic positions of lincRNAs were extracted from the NONCODE database (v5.0) for human and bovine.Target prediction and functional enrichment analysis Typically, lncRNAs regulate their target genes through cis or trans modes. The cis-acting refers to the regulation of genes in a host (in the case of ilncRNAs) or nearest neighboring protein coding genes (in the case of lincRNAs). According to the location of the lncRNAs and mRNAs on the chicken genome, 10/100 kb upstream or downstream of the differentially expressed lncRNAs were searched to identify the nearest neighboring protein coding genes, which can be considered as potential target genes of these lincRNAs20. On the other hand, trans-acting refers to the influence of lncRNAs on other genes at expression levels, which can be assessed by co-expression analysis (correlating expression levels between lncRNAs and mRNAs)14. To predict the potential target genes of the lncRNAs in trans mode, Pearson correlation coefficient was calculated based on the expression levels of each pair of lncRNA and mRNA (r > 0.95 or r <  − 0.95). Functional enrichment analysis including Gene Ontology (GO) and KEGG pathway analysis48 related to the predicted target genes of lncRNAs was conducted using the EnrichR database49. A term with an adjusted P-value < 0.1 (FDR) was considered to be significantly enriched.QTL mapping analysis of LncRNAs The co-localization analysis was performed to determine if the putative novel lncRNAs are located in the QTLs related to feed efficiency. For this purpose, all QTLs related to feeding efficiency were retrieved from AnimalQTLdb50. Then positions of the lncRNAs were compared to the QTL's locations.Target prediction of the candidate LncRNAs and PPI network The potential protein–protein interaction (PPI) network was constructed for the predicted cis and trans target genes of the lincRNAs and ilncRNAs (known and novel) using STRING database . 51Then, Cytoscape software (version 3.7)52 was applied to visualize and combine the significant interactions obtained by STRING along with predicted cis and trans target genes of lncRNAs (separately for each of lincRNAs and ilncRNAs). The key sub-networks (module genes) were detected using a Cytoscape plugin, ClusterONE tool (version 1.0), based on minimum size = 5 and a minimum number of the genes in a module = 553. In order to further explore the characteristics of the significant modules, functional enrichment analysis was performed on their gene members.To investigate various aspects of the putative lncRNAs, five different analyses were performed based on our previous study, which is described as follow54. The details of all the designed primers are provided in Supplementary File S1. RT-qPCR was performed on a Light Cycler 96 instrument (Roche Co. Germany) using HiFi SYBR Green Master Mix . RT-qPCR was performed with the following thermo cycling conditions: an initial 1 cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, 60 °C for 20 s and 72 °C for 20 s, followed by a 72 °C elongation for 60 s. GAPDH and ACTB were used as house-keeping genes to normalize the expressions of lncRNAs55. The results were analyzed using the 2 − ΔCt method.To validate the RNA-Seq results by RT-qPCR, seven differentially expressed lncRNAs (including four known lincRNAs and three novel lincRNAs) were randomly selected. Two biological replicates were conducted for each breed. Total RNA was extracted from the liver of chickens using TRIzol  according to the manufacturers’ protocols and the concentration and purity of RNAs were measured using NanoDrop 2000. The cDNA was synthesized from purified RNA by using an RT-qPCR kit . Primer 3 plus software was applied to design the primersAll methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Animal Care and Use Committee at the Animal Science Research Department, Safiabad Agricultural.In our previous study, the average BWG of higher RFI and lower RFI groups had obtained 441.06 ± 6.76 g and 1027.88 ± 7.57 g, respectively. The significant differences (p < 0.01) in daily feed intake (DFI) had observed between native and commercial chickens (1329.25 ± 8.05 vs 2007.90 ± 9.25). The lower RFI had shown a higher average daily weight gain than a higher one. Consequently, the differences in mean RFI values between LRFI and HRFI chickens in each breed were highly significant (p < 0.01). The local breed had the RFI values of 13.430 ± 5.393 (g/day) compared with –11.212 ± 4/435 (g/day) for the commercial breed during 19 days (day 24–42) of the experiment".A total of 35,696,493 and 34,824,326 raw paired-end reads were obtained in native and commercial breeds, respectively. Following a pipeline of adaptor removal, quality filtering and the removal of sequences that were too short, only 565,714 and 534,690 paired reads did not pass the quality filtering and were removed in native and commercial breeds, respectively. In total, 84.70–86.97% of the clean reads from all the samples were successfully aligned to the reference genome. Of the successfully aligned reads, 82.90–84.53% were uniquely aligned , which are not annotated in the chicken GTF file (ENSEMBL database), yet. Finally, according to our stringent lncRNA prediction pipeline, 587 putative novel lncRNAs including 454 lincRNAs and 133 ilncRNAs were identified , which is lower than known mRNAs (50%) , followed by chromosome 2 (72 lncRNAs), 3 (71 lncRNAs), 5 (51 lncRNAs) and Z (43 lncRNAs), whereas chromosomes 22, 23 and 25 contained only ilncRNAs , respectively  and human lncRNAs. In addition, of 16 novel and nine known conserved lincRNA transcripts, eight and two genes showed evidence of sequence homology among bovine, human or mouse (Supplementary File S2). Overall, transcript-level homology of our predicted lncRNAs with active transcribed lncRNAs in human, cow and mouse detected, and found that only 2% of known and 3% of novel lncRNAs can be aligned to investigated species. In agreement with our results, it was reported that less than 1% of chicken lincRNAs owned detectable sequence conservation with human or mouse lincRNAsThe results of the synteny analysis indicated a similar structural architecture between the performed comparisons. Results of this analysis revealed that 62 known and 96 novel lincRNAs as well as 89 known and 116 novel lincRNAs were located in the neighborhood of respective orthologous protein coding genes in chicken/cow and chicken/human comparisons, respectively (Supplementary File S3). Out of 62 and 96 lincRNAs, two and three lincRNAs had conserved sequencing with bovine and human, respectively. Also, two known and four novel lincRNAs, out of 89 and 116 syntenic genes, had an orthologous sequence in bovine and human, respectively.38.Moreover, 121 known lincRNAs and 212 novel lincRNAs were determined with conserved synteny among the investigated species. Of these, 26% (31 of 121) of known lincRNAs and 23% (48 of 212) of novel lincRNAs showed sequence homology with the investigated species. These findings were consistent with the previous study that reported higher conservation in synteny of the lncRNAs in comparison to their sequencesIn total, 17,833 transcripts  were identified to be expressed in both breeds. Of these, 1,040 and 1,081 transcripts were expressed only in commercial and native breeds, respectively. Therefore, 15,712 transcripts were commonly expressed in both breeds. To identify potential feed efficiency related genes, differential expression analysis was applied and FPKM values of the genes were compared between the two chicken breeds. The analysis showed that 39 known lncRNAs  and 87 known mRNAs (including 63 up- and 24 down-regulated) were DEGs. In addition, 14 novel lincRNAs (four up and 10 down-regulated) tended to be differentially expressed. No annotated lncRNA and novel ilncRNAs were identified to be differentially expressed. The top five differentially expressed lncRNAs in each lncRNA class are represented in Table To further confirm the accuracy of the sequencing data, RT-qPCR was performed on seven differentially expressed lincRNAs. As shown in Fig. To understand the biological functions of the lncRNAs, cis-regulated genes of these genes were investigated. The genes transcribed in the 10/l00 kb upstream or downstream around the lincRNAs are generally considered to be cis target genes. All 52 (38 known and 14 novel) differentially expressed lincRNAs were detected to be located near of 70 mRNAs and these mRNAs were involved in "lipid metabolism", "carbohydrate metabolism", "growth" and "cell death".Trans target genes of the known and novel lncRNAs were predicted through expression correlation analysis. In total, 53 lincRNAs (48 known and five novel lincRNAs) were corresponded to 619 target genes (Supplementary File S5). The identified co-expressed genes were similar for the most of known lincRNAs. Accordingly, lincRNAs with similar target genes were clustered, which led to four clusters. The first, second, third, and fourth clusters included 4, 3, 15, and 24 lincRNAs, which were co-expressed with 179, 170, 133, and 130 target genes, respectively. Functional enrichment analysis of the target genes in cluster number one and two were significantly enriched in one and three GO terms and one and three KEGG pathways, respectively, which were related to biological processes such as “gamma-aminobutyric acid signaling pathway”, “ear morphogenesis”, “cochlea morphogenesis” and “inner ear morphogenesis”. Also, nine and 29 significant GO terms were found in clusters number three and four, respectively. The detailed information related to these clusters and their functional analysis are provided in Supplementary File S6. According to the previous studies, feed efficiency-related terms in clusters number three and four were detected including “monocarboxylic acid transport”, “potassium ion transport”, “chloride ion homeostasis”, “negative regulation of calcium ion trans membrane transport” and “cellular response to cAMP”. KEGG pathway analysis was further performed and no significant pathways were identified.In ilncRNAs, 10 known ilncRNAs were corresponded to 265 target genes (Supplementary File S5). Based on the co-expressed genes of the known ilncRNAs, which were similar among the ilncRNAs, two clusters were constructed. Three ilncRNAs were expressed in cluster number one with 130 target genes. Also, there were four ilncRNAs corresponding to 130 target genes in the second cluster. Interestingly, GO analysis of these target genes led to detection of 38 enriched terms (nine GO terms in cluster one and 29 GO terms in cluster two)  were connected to 1,514 mRNAs with 11,160 connection edges. Moreover, a network with 216 ilncRNAs (68 known and 168 novel) and 416 protein-coding genes along with 1,490 interactions were obtained. ClusterONE tool predicted one significant module in each of the networks with < 20 nodes including green module  were significantly enriched in 165 GO terms and seven KEGG pathways, which were related to glucose homeostasis (GO:0042593), calcium ion transport (GO:0006816), insulin receptor signaling pathway (GO:0008286), lipid phosphorylation (GO:0046834). The light-blue module (ilncRNA-based network) were enriched in 132 metabolic pathways and four KEGG pathways including “positive regulation of ion transport”, “positive regulation of sodium ion trans membrane transporter activity”, “negative regulation of lipid metabolic process” and “carbohydrate homeostasis”. The functional enrichment analysis of the two modules showed that both are related to feed efficiency (Supplementary File S7).58. These genes can be considered as promising candidate genes responsible for feed efficiency in chicken . Of these, ilncRNA.2932 and lincRNA.5494, which were located in feed intake related QTLs, were specifically expressed in commercial breed. Out of 47 novel lincRNAs, one differentially expressed lincRNA (lincRNA.10349) was overlapped with a QTL related to residual feed intake (RFI). Moreover, seven lincRNAs  were located in more than one QTL related to feed efficiency (Supplementary File S8). Interestingly, several cis target genes of these lincRNAs, including KCNRG, DLEU7, CTSC and RAB30, were reported to be involved in feed efficiency and RFI or has a known role the growth rate 60. Whereas many of the lncRNAs and their functions are known in different species, such as humans and mouse, lncRNA research is in its infancy in domestic animals, especially in chickens61. In addition, the role of lncRNAs in the liver of chicken in regulating feed efficiency-related traits is unclear. The most common way of identifying relevant lncRNAs is by differential expression between contrasting conditions38. Hence, several potential known and novel lncRNAs were identified in the present study by comparing the gene expression profile of the liver of two extremely different chicken breeds (Iranian native chicken vs Ross breed). Comparing the features of the identified novel lncRNAs to protein coding genes showed consistency between our results and the previous studies, as lncRNAs had a lower expression, shorter transcript length62, fewer exons63 and lower GC content relative to mRNAs64. These findings indicate that the used bioinformatic pipeline is reliable. Only 2% of known and 3% of novel lncRNAs showed transcript-level homology with known lncRNAs in human, cow and mouse was, which is in agreement with Kuo et al.37. In this regard, Liu et al. in their study reported that less than 1% of chicken lincRNAs owned detectable sequence conservation with human or mouse lincRNAs and confirmed that lncRNAs in vertebrates have low levels of interspecies short and highly conserved regions17. Also, in agreement with our previous study, synteny and conservation analysis of the novel lncRNAs emphasized that synteny of lncRNAs is more conserved than their cross-species sequence conservation38. Therefore, syntenic analysis can be considered as a useful approach for improving the prediction of novel lncRNAs in the genomes that are incompletely annotated. In brief, 1,110 annotated lncRNAs, 525 known lincRNAs, 68 known ilncRNAs, 454 novel lincRNAs and 133 novel ilncRNAs were found. Of these, 38, 1, and 14, known lincRNAs, known ilncRNAs and novel ilncRNAs, respectively, were identified as DEGs. Although differentially expressed genes can be considered as potential candidates related to feed efficiency, however further investigations are needed to be ensured about their potential roles in these biological processes. Hence, different functional analysis was applied to further understand the functions of these genes in regulating feed efficiency in chicken.Recently, lncRNAs have received much consideration and a growing number of studies have shown that lncRNAs play key roles in various physiological and pathological processes65. Accordingly, in the cis mode, the identified target genes of the novel lincRNAs were mainly involved in lipid metabolism  and carbohydrate and growth metabolism, which are related to feed efficiency and make these novel genes as ideal candidates to investigate the regulatory mechanism of feed efficiency in chicken. The relationship between lipid metabolism and feed efficiency has been reported in previous studies, as animals with lower feed efficiency have higher fat deposition and cholesterol levels67. One of these target genes was ethyl malonyl‑CoA decarboxylase , which is a new metabolite proof reading enzyme. ECHDC1 can eliminate ethyl malonyl‑CoA by converting it to butyryl‑CoA68. Interestingly, ECHDC1 was a DEG in our study that was up-regulated (fold change = 0.52) in Ross breed and showed an opposite expression pattern with its neighboring lincRNA.103491.1. Moreover, these genes were located in the QTL regions associated with feed efficiency, which reinforce their potential function in this subject. These findings make these genes (lincRNA and its target gene) as interesting candidates for a follow-on experiment to assess their impact on feed efficiency in chicken. Lipolysis proceeds from the hydrolytic cleavage of ester bonds in triglycerides (TGs), and result in the generation of fatty acids (FAs) and glycerol69. Increased lipolysis of fatty tissues may increase circulating fatty acid concentrations and displacing the muscle's preference for fatty acid oxidation70. Moreover, excessive fat accumulation has a negative effect on feed efficiency71. VPS13C, a member of the VPS13 family of proteins , regulates galectin-12 stability in adipocytes72, was predicted to be targeted by a novel lincRNA.2454.1 and its target gene was up regulated (fold change = 0.17) in high efficient chickens . In this regard, the important roles of galectin-12 in adipocyte differentiation and lipolysis for the regulation of whole-body adiposity have been reported72. In addition, VPS13C is associated with regulation of glucose homeostasis72. Lemley et al. in their study suggested this gene to play an important role in glucose homeostasis for high milk production in dairy cow73. Therefore, the glucose homeostasis process probably stimulated lipolysis process74, indicating that high efficient chickens have a lower amount of abdominal fat75.This finding also emphasized the functional diversity of lncRNAs, which may contribute to widespread regulatory roles in the liver tissue of chicken. The membrane-bound PRLR, as a member of the cytokine receptor family, is closely related to the growth hormone receptor. Over 300 separate biological activities have been attributed to PRL including endocrine signaling, metabolism, control of water and electrolyte balance, growth and development76. PRLR is predicted to be a target gene of lincRNA.15581.1 and its higher expression in commercial chicken (fold change = 0.98) can increase feed efficiency by increasing growth rate and decreasing storage energy77.Recent studies suggested that the function of lncRNAs can be deduced by analyzing their co-expressed mRNAs or neighboring protein-coding genes in genome wide78 and cholesterol biosynthesis79, were known to be associated with feed efficiency. The ACAA2 gene that was predicted to be targeted by lincRNA.15325 implicated in mitochondrial fatty acid oxidation. ACAA2 encodes an enzyme that catalyzes the cleavage of 3-ketoacyl CoA to yield acetyl-CoA and acyl-CoA, the final step of the mitochondrial fatty acid beta-oxidation spiral. The high expression level of ACAA2 was found in the muscle of the high feed efficient birds78. Furthermore, a previous study by focusing on growth traits and important metabolic pathways in pigs reported ACAA2 as a candidate gene involved in fatty acid metabolic, which is an essential source for energy80. Accordingly, high efficient chicken had higher expression of genes involved in fatty acid uptake and oxidation78. The up regulation of lincRNA.15325 and its related target gene (fold change = 0.13) in the commercial breed may lead to higher fatty acid metabolism to supply energy. Cholesterol is an essential component of animal cell membranes that gives cell membranes elasticity and fluidity and is involved in cellular signaling and intracellular transport82. It is reported that an increase in the expression of EBP, a key gene functioning in cholesterol biosynthesis, exhibited greater rates of gain and feed efficiency of beef cattle during compensatory growth83. Moreover, divergent genes related to cholesterol biosynthesis were upregulated in high efficient chicken78. Interestingly, consistent with the above findings the high efficient group had a higher expression level of lincRNA.2988 (fold change = 2.02) and its target gene (fold change = 0.035). B3GALT5  was predicted as a cis target gene of lincRNA.1424. B3GALT, as an O-glucosyltransferase, participate in the elongation of O-fucosylglycan and is involved in fucose metabolic pathway, which is a part of innate immunity glycoproteins (mucins)84. Fucose has kept the integrity of the mucosal barrier and has made a relationship between the host tissue and intestinal microbiome in studies related to mice85 and rabbits86. The fucose metabolism process was positively related to feeding efficiency in bovine, which is reported by Roehe et al. 87. Moreover, B3GALT was identified as DEG in Zeng et al. study that conducted a study to identify duodenum genes and pathways through transcriptional profiling in two extreme RFI phenotypes of the duck population61. Therefore, overexpression of lincRNA.1424 and B3GALT (fold change = 1.42) in the commercial breed may contribute to the development of the microbiome in the intestine. Also, lincRNA.14534, lincRNA.313, lincRNA.15325, lincRNA.5912, lincRNA.6167, lincRNA.1460 and lincRNA.2104 were up-regulated in the liver of high efficient breed (Ross) and their expression were positively correlated with LRRC8D, LRRC8C28, LMAN188, CTDSPL57, CDCA7L89, CHST790 and AGPAT391. Previous studies reported these target genes to be located within the most significant SNPs associated with RFI or feed efficiency.The genes that were identified as targets of differentially expressed known lincRNAs and also were associated with efficiency-related traits were included B3GALT5, ACAA2, CDCA7L, CHST7, LRRC8D, LRRC8C, LMAN1, CTDSPL, EBP, and AGPAT3. Some biological processes like lipid metabolism92. G protein-coupled receptor (GPCR) family as members of signaling pathways has a crucial role in feeding behavior and energy utilization93. Melanin-concentrating hormone receptor (MCHR1) binds MCH with high affinity and the orphan GPCR is a receptor for MCHR194. Therefore, MCHR1 is capable of stimulating various signaling pathways particularly triggering of MAPK/ERK signaling cascade and increase the calcium concentration95. Moreover, it has been suggested that MCH might play important roles in the CNS of vertebrates, particularly in the regulation of energy balance in chicken96. Therefore, it can be speculated that the overexpression of MCHR1 and its sixteen co-expressed lincRNAs might integrate signals through CNS and peripheral tissue and modulate feeding and energy expenditure, as well affect feed conversion ratio. ADRA2A  is a component of the GPCR superfamily and has an important role in the regulation of neurotransmitter release from adrenergic neurons and sympathetic nerves in the central nervous system98. ADRA2A is involved in different processes of the neurotransmitter, blood pressure, central nervous system and lipolysis99. Moreover, ADRA2A is reported to be involved in the fat metabolism pathway. However, the reported SNPs of this gene are associated with increased glucose levels100,101. Mărginean et al. in their study by investigating the ADRA2A gene polymorphisms and mothers–infants' nutritional status, found that defects in the ADRA2A gene might have a dichotomous role, leading to the development of obesity or underweight101. Also, a significant lower feed conversion ratio (FCR) was observed in lambs with the CC genotype of the ADRA2A g.1429 C > A mutation rather than those carrying the AA and CA genotypes 102. Therefore, ADRA2A by increasing glucose level and fatty acids, probably implicates the lipid metabolism process and energy metabolism. It is well known that lipid and energy metabolism are significant processes in the feed conversion rate of poultry and livestock104. Zhang et al. by focusing on candidate genes and pathways related to feed efficiency in Hu sheep, reported ADRA2A as candidate gene in feed efficiency102. In consistent with these findings, Zhang et al. in their study reported ADRA2A as DEG in the liver of Hu sheep with different residual feed intake105. In the present study, ADRA2A was overexpressed in commercial animals and fifteen lincRNA genes were predicted as potential regulators of this gene, in trans mode.Sixteen lincRNAs were found to be co-expressed significantly with the MCHR1 gene. Central nervous system (CNS) through hypothalamic neural circuits integrates peripheral signals related to energy and nutrient status and interprets those to coordinate between feeding behavior and energy expenditure. The previous studies reported that CNS contributes to feed intake regulation with peripheral tissue mechanisms106. Gamma-Aminobutyric acid (GABA) is found in the hypothalamus of birds and mammals and functions as a significant inhibitory neurotransmitter107. Several studies have found that stimulating of GABAergic system led to increased food intake in bird and mammals109. In this regard, GAD2 has found as DEG in a transcriptome analysis of hypothalamus-regulated feed intake20. In addition, GAD2 gene variants had significant associations with average daily feed intake (ADFI) in beef cattle110. Accordingly, it probably contributes to increase feed conversion ratio and make a balance between feeding and energy expenditure through hypothalamus. As mentioned above, the predicted target genes for the known lincRNAs were similar to the known ilncRNAs, which indicate a synergistic effect between lincRNAs and ilncRNAs for regulating a common biological process. LincRNA.13441 (as a novel lincRNA) was predicted as a potential regulator of 133 genes. Its target genes were enriched in various biological categories including “calcium ion transport” and “muscle contraction regulation”. It is well documented that calcium signaling is an important modulator of lipid metabolism111. Hence, the function of these lincRNAs (such as ADRA2A and MCHR1) could be closely related to lipid metabolism as well as feed efficiency development due to their co-expressed targeted mRNAs in the commercial breed affect lipid metabolism.The other known lincRNA (lincRNA.14916) was found to target GAD2, which is associated with stimulating food intake. LincRNA.14916 and its co-expression gene were up-regulated in the commercial breed. The GAD2 gene is responsible for encoding the glutamic acid decarboxylase enzyme (GAD65) and catalyzing the constitution of Gamma-aminobutyric acid (GABA) from L-glutamic acid112. The lipid and energy metabolism were known as significant pathways related to feed conversion rate of poultry and livestock104. Moreover, this gene was previously found as a significant DEG in sheep with different feed efficiency105. Furthermore, as discussed earlier, MCHR1 as a receptor for GPCR participates in feeding behavior and energy utilization94. These results indicated that the identified lncRNAs might play regulatory roles in the feed efficiency-related terms.To better understand how lncRNAs cooperate with their target genes, integrated networks (separately for each of lincRNAs and ilncRNAs) were constructed and one significant module was found in each of the networks. Member genes of both modules were significantly enriched in pathways related to "regulation of lipid catabolic process" (such as ADRA2A) and "translating signals" (such as MCHR1). ADAR2A encodes adrenoceptor alpha 2A and is a regulator of catecholamines, which have been introduced to be associated with energy metabolism and fat metabolism68, lincRNA.10336 (target SMAD3)113, lincRNA.2986 (target EBP)83 and lincRNA.10372 (target TPD52L1)114 were related to lipid metabolism. For example, SMAD3 is a multifaceted regulator in adipose physiology, pathogenesis of obesity and type 2 diabetes113. As discussed above, animals with lower feed efficiency have higher fat deposition and cholesterol levels67. In addition, six lincRNAs  were located in more than one QTL related feed efficiency. CTSC, a predicted cis target gene of lincRNA.2201, encodes a lysosomal cysteine proteinase and play a central role in bacterial killing and immune regulation in T lymphocytes. The effects of this gene on the observed difference between the pigs with low and high RFI have been reported58. The closest protein coding gene to lincRNA.1964 was DLEU7, which encodes a protein containing 221 amino acids. Fibroblast growth factor (FGF) regulates the expression of DLEU7 during early embryogenesis56. Moreover, an association of this gene with human height has been reported117, which can be suggested to play an important role in chicken growth and feed efficiency. The other gene related to growth rate was RAB30 (cis target of lincRNA.2215). Claire D' Andre et al., conducted a study on the identification and characterization of genes that control fat deposition in chicken118. RAB30 was appeared to be down-regulated in slow-growing Xinghua chickens. lincRNAs that were located in QTL regions related to feed efficiency compared with other lincRNAs are more likely to be truly related to feed efficiency. Cis target genes of these lincRNAs that were involved in feed efficiency and RFI or has a known role in the growth rate and lipid metabolism, were interesting functional candidate genes responsible for feed efficiency in chicken. Our findings supported this hypothesis that lncRNAs may affect feed efficiency mainly through regulating lipid metabolism, glucose homeostasis, growth rate, immune system, modification of proteins and energy homeostasis. However further experiments still require to validate the suggested functions of these lncRNAs.QTL analysis revealed that 11 novel ilncRNAs and 47 novel lincRNAs as putative effective lncRNAs in feed efficiency related processes, as they were overlapped with the potential regions associated with RFI in genome wide. Of these, the predicted target genes of the four lincRNAs including lincRNA.10349 (target ECHDC1)The present study provided a valuable resource to clarify the genetic basis of feed efficiency and further experiments will corroborate the function of all the RNAs the reported here. To do this end, RNA-Seq data along with bioinformatics approaches were applied and a series of lncRNAs and target genes were identified. Our results indicated that some of the lncRNAs might regulate locally their neighboring genes in cis mode as well as in trans mode. Functional enrichment analysis showed that the identified lncRNAs had enough potential to be related to feed efficiency, as their predicted target genes were significantly involved in the biological processes and KEGG pathways associated with feed efficiency including lipid metabolism, carbohydrate metabolism, and growth. Moreover, several lncRNAs were identified that were overlapped with QTLs controlling feed efficiency such as RFI, which highlighted their importance in this context. Although little is known about the functions of lncRNAs in chicken, our results provided the initial step for studying how changes in lncRNA expression affect the regulation of mechanisms involved in chicken feed efficiency.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.


On the rise due to its effects of euphoria, recreational use of nitrous oxide can cause severe neurologic symptoms caused by marked and rapid onset of vitamin B12 deficiency. This is a case of a young 34-year-old male who presented with an inability to walk and confusion caused by vitamin B12 deficiency in the setting of recreational nitrous oxide use. Recently, the recreational use of nitrous oxide has been increasing due to its euphoric effects . Many obA 34-year-old male with a history of alcohol use and dependence comes in by ambulance for generalized weakness. The patient is oriented to himself only, confused, and unsure why he is in the hospital. The patient’s father states he cleaned out 40,000 used nitrous oxide cartridges from the patient’s apartment, which he has used over the last seven to eight weeks (averaging use of >600 whippets/day). Vital signs are stable: blood pressure of 113/70 mmHg, heart rate of 95 bpm, respiratory rate of 18 breaths/minute, 98% pulse oximetry, the oral temperature of 37.4 degrees Celsius. Laboratory assessment shows mild macrocytic anemia  present with weakness, ataxia, and ascending paresthesia ,5,6. In If normal vitamin B 12 levels are detected, it is important for providers to check methylmalonic acid and homocysteine levels-they will be elevated . Early rIt is vital for healthcare providers to recognize the connection between nitrous oxide use and vitamin B12 deficiency to timely supplement patients to relieve neurological deficits. Whether it is during an evaluation for vitamin B12 deficiency or nitrous oxide use, the provider should be aware of their connection.


Catestatin (CST) is an important peptide that influences various inflammatory diseases. Our goal was to investigate CST concentrations in patients with RA compared to healthy subjects. This cross-sectional observational study included 80 patients with RA and 80 healthy control subjects. Demographic characteristics and laboratory parameters were recorded. Serum CST levels were determined by an enzyme-linked immunosorbent assay (ELISA). Serum CST levels were significantly higher in RA patients than in the control group . In RA patients, there was a statistically significant correlation between CST and patient age  and both DAS28  and HAQ scores . There was a statistically significant correlation between serum CST levels and RA duration . Multiple linear regression analysis showed that serum CST levels retained a significant association with RA duration  and DAS28 score  after model adjustment for age, body mass index (BMI) and HAQ score, with serum CST levels as a dependent variable. These findings imply that CST is possibly associated with RA complex pathophysiology and disease activity. However, future larger multicentric longitudinal studies are necessary to define the role of CST in RA. RA is characterized by progressive joint damage leading to physical disability and is associated with various extra-articular manifestations. It is considered to be caused by a combination of genetic and epigenetic components, abnormalities in the cellular and humoral immune response and exposure to environmental factors 2. Chronic systemic inflammation in RA has a key role in accelerated atherogenesis through direct and indirect effects on the vascular endothelium and myocardium, contributing to higher cardiovascular (CV) risk in addition to classical CV risk factors such as obesity, smoking, dyslipidemia and hypertension6.Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease of unknown etiology that affects 0.5 to 1% of the general population10. According to previous studies, CST has a significant role in the reduction of endothelial inflammation7, regulation of blood pressure12, suppression of atherosclerosis and inhibition of coronary vasoconstriction13. CST is believed to have an important role in the pathophysiology of heart failure, acting as an attenuator of the cardiac inflammation through shifting differentiation of macrophages to more anti-inflammatory phenotypes15. Moreover, CST is particularly of interest because of its immunomodulatory role, including metabolic regulation and immune homeostasis16. Recently published studies demonstrated significantly increased serum CST levels in patients with inflammatory bowel disease (IBD), which is in concordance with in vitro findings of the impact of CST on intestinal microbiota19. Those findings have implicated a possible contribution of CST to higher CV risk, as well as its role in anti-inflammatory responses in these patients19. Interestingly, it was proposed that circulating CST in human plasma could be intermediate hereditary phenotype for increased CV risk20.Catestatin (CST) is a multifunctional bioactive peptide that is proteolytically cleaved from chromogranin A (ChgA). It primarily acts as an inhibitor of catecholamine secretion and as a stimulator of histamine release22. However, elevated serum levels of proinflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukins-1 and -6 , and autoantibodies contribute to systemic inflammation, which makes RA a major independent CV risk factor24. Therefore, management of both traditional risk factors and RA disease activity should be imperative22. Moreover, recent studies have implicated a potential clinical application of CST and other CgA cleavage products as diagnostic markers, as higher concentrations of these peptides have been detected in patients with CV diseases (CVDs), IBD, chronic kidney disease and RA26. As already mentioned, several recent studies have shown that CTS attenuates the inflammatory response through the downregulation of macrophage differentiation toward the production of proinflammatory cytokines 28. Therefore, all of the aforementioned evidence points to a possible link between RA and CST through either an immunomodulatory or atherogenic role. To the best of our knowledge, until this point, there have been no published studies that explored the association of serum CST levels with RA.Previous studies demonstrated that the high prevalence of classical CV risk factors does not fully explain the 50% increased risk of CV-related morbidity and mortality in RA patientsThe aim of this study was to determine serum CST levels in RA patients in comparison with healthy controls and to assess the relationship between CST levels and chronic inflammation, duration and activity of RA, as well as other clinical, biochemical and anthropometric parameters.This cross-sectional observational study was conducted at the Department of Internal Medicine, Division of Clinical Immunology and Rheumatology, University Hospital of Split over a period from October 2020 to June 2021.The study was conducted according to the guidelines of the Declaration of Helsinki of 1975, as revised in 2013, and approved by the Ethics Committee of the University Hospital of Split . All subjects were informed about the procedures and purpose of the study in a timely manner. Informed consent was obtained from all subjects involved in the study.29. All participants were above 18 years of age and underwent the full study protocol, except measurement of disease activity [using Disease Activity Score-28 (DAS28) and functional disability [using The Stanford Health Assessment Questionnaire (HAQ)], which were measured only in the patient group32. Patients with RA were recruited from the outpatient clinic of the Department of Clinical Immunology and Rheumatology, University Hospital of Split, while control subjects were recruited from healthy blood donors, medical staff and healthy volunteers from the local primary health care center.This study included 80 patients with RA and 80 healthy control subjects matched by age and sex. One hundred RA patients were screened for inclusion in the study, and 85 patients were eligible for inclusion in the study. Among the participants mentioned, three were excluded due to refusal of further procedures, and two were excluded due to sudden disease worsening. A total of 120 healthy control subjects were screened at the beginning of the study. Among them, twenty participants were excluded due to inability to schedule an appointment and remaining others due to refusal of further procedures. RA diagnosis was made in accordance with the latest 2010 American College of Rheumatology/European League Against Rheumatism Classification Criteria for RAInclusion criteria were: disease duration of at least 2 years; age between 18 and 75 years; mandatory treatment with disease-modifying antirheumatic drugs (DMARDs), including biologic DMARDs (bDMARDs) or targeted synthetic DMARDs (tsDMARDs): TNF inhibitors (TNFi’s) which includes adalimumab, certolizumab, etanercept, golimumab or infliximab, B-cell targeted therapies (rituximab), IL-6 inhibitors (sarilumab or tocilizumab) and Janus kinase (JAK) inhibitors .2]; history of heart failure; respiratory disease; liver disease; active malignant disease; clinically significant drug abuse and consumption of alcohol more than 40 g/day, and long-term exposure to oral glucocorticoid therapy ≥ 10 mg/day.The exclusion criteria were: chronic inflammatory disorders other than RA; diabetes mellitus; pregnancy; breastfeeding; renal impairment . We excluded all participants who showed any sign of inflammation in any of these steps.2)]. Arterial blood pressure was measured using a standard mercury sphygmomanometer Riester Big Ben Aneroid  with a suitable arm, and values were obtained after a minimum of two measurements. The subjects were seated and relaxed for 10 min before the measurements, with the upper arm at heart level. The mean values of arterial blood pressure was determined after those two measurements. Data on smoking, coffee and alcohol consumption were taken from all study participants. Finally, we checked the medical records of all patients and extracted clinical data relevant for this study.All participants had a detailed medical history, a physical examination and anthropometric data assessment. Anthropometric measurements included body height, weight and body mass index (BMI). A calibrated medical scale with built-in heights  was used to measure body mass and height, and BMI was calculated according to the formula = [body weight (kg)]/ plus measure of general health . Disease activity was graded according to the following thresholds: DAS-28 score < 2.6 indicates remission; score from 2.6 to 3.2 indicates low disease activity; score from 3.2 to 5.1 indicates moderate disease activity and; DAS-28 score above 5.1 is considered high disease activity31.DAS score in RA patients was performed by two experienced rheumatology specialists independently and score was estimated using the online Dawn32.The HAQ was used to examine the extent of functional ability in daily living and activities in patients with RA. The HAQ score ranges from 0 to 3 and is a good predictor of future disability. HAQ was performed by an experienced rheumatology specialist. The HAQ evaluates the patient’s level of functional ability through 20 questions in eight categories, which include the patient’s disturbance of movements and activities that involve both upper and lower extremities. In accordance with previous findings, a score below 0.5 is considered normal, whereas a score above 1.5 indicates severe disability®  and MedCalc . Continuous data are presented as the mean ± standard deviation or median (interquartile range) based on the variable distribution normality, whereas categorical data are expressed as numbers (N) with percentages (%). Normality of distribution for continuous variables was assessed using the Kolmogorov–Smirnov test. For differences between the groups, an independent samples t-test was used for continuous variables with normal distribution, whereas the Mann–Whitney U test was employed for the analysis of continuous variables with non-normal distribution. The chi-squared (χ2) test was used to determine differences between groups in terms of categorical variables. To investigate the correlation between CST and various clinical parameters, we used either Pearson’s correlation coefficient or Spearman’s rank correlation coefficient based on the variable distribution normality. Comparison of serum CST levels between RA duration tertiles was performed using one-way analysis of variance (ANOVA) with a post hoc Tukey test. Finally, a multiple linear regression analysis with a forward algorithm was applied to determine significant and independent correlates of the CST serum levels, which was defined as a dependent continuous variable. The level of statistical significance was set at a p-value < 0.05.The data for the present study were analyzed using SPSS Statistics for WindowsSample size analysis was conducted using the data from a pilot study on 10 subjects from the RA population and 10 matched control subjects. The value of serum CST, which was the main result of the study, was used for the calculation. The mean serum CST levels were 9.40 ± 2.75 ng/mL in the RA group and 3.71 ± 2.55 ng/mL in the control group. With a type I error of 0.05 and a power of 90%, the required sample size was 29 participants per group.There were no statistically significant differences in anthropometric measurements between the RA patients and the healthy control group. The demographic and anthropometric characteristics of the RA patients and the healthy control group are shown in Table In the laboratory analyses, the RA group had significantly higher levels of serum creatinine , hsCRP , total cholesterol  and LDL (3.2 (2.5–3.7) vs 2.8 (2.1–3.6) mmol/L, p = 0.034). Moreover, the RA group had a significantly lower level of 25OHD   . There were no other statistically significant correlations between catestatin levels and laboratory or anthropometric parameters  and HAQ score    showed significantly higher levels of serum CST in the 3rd tertile (12.99 ± 3.60 ng/mL) than in both the 2nd (9.70 ± 2.81 ng/mL) and 1st (8.87 ± 3.97 ng/mL) tertiles (p < 0.05)  and DAS28  after model adjustment for age, BMI and HAQ score, with serum CST levels as a dependent variable  and consequent changes in the inflammatory environment13. VCAM-1 is a glycoprotein expressed by endothelial cells that mediates leukocyte-endothelial cell adhesion during inflammation41. Several studies reported significantly higher serum VCAM-1 levels in patients with RA44. In addition, serum VCAM-1 levels correlate with endothelial dysfunction and the initiation and progression of RA45. The expression of VCAM-1 in synovial tissue is upregulated by proinflammatory cytokines (TNF-α and IL-6)45. By lowering the production of proinflammatory cytokines, CST could decrease the expression of adhesion molecules in the endothelium. Moreover, an in vitro study showed that anti-VCAM-1 monoclonal antibody injection into a collagen-induced RA mouse model did not alter the incidence, but decreased the severity of arthritic joints compared with the control mice46. Another possible mechanism by which CST regulates macrophages refers to the polarization of RA synovial tissue “activated macrophages” towards two different phenotypes depending on the disease activity: M1  and M2 47. The M1 phenotype releases high levels of proinflammatory cytokines and consequently prevails in patients with highly active RA alongside the increased presence of proinflammatory macrophage receptor with collagenous domain (MARCO)48. Based on the results of in vivo and in vitro studies, CST treatment decreases proinflammatory cytokine  and macrophage levels40. Additionally, the production of CST by macrophages could result in reduction of the inflammation by feedback inhibition mode15. Considering that the RA population investigated in this study was treated with biological agents, a synergistic effect of those drugs and CST in RA is possible. Although this could, to some extent, explain the association between CST and RA, further studies are necessary to confirm the precise role of CST in the complex pathophysiology of RA.In addition to higher CST levels in our group of RA patients than in healthy subjects, we found that CST is an independent predictor of disease activity and functional disability. Based on the existing literature, a possible explanation for these findings is that CST participates in a cascade of signal transduction pathways between immune and noimmune cells, particularly altering the balance between pro- and anti-inflammatory cytokines49. Moreover, CVDs are still the leading cause of mortality in patients with RA (30–40% of deaths)51. Additionally, it has been established that both traditional CV risk factors and intrinsic RA features contribute to excess CV-related morbidity and mortality22. The most significant intrinsic RA features are the disease duration, the presence of ACPAs, RF, elevated proinflammatory cytokines and hsCRP serum levels57. Premature atherosclerosis is the hallmark of CVD development in RA3. According to this point, the precise effects of CST on atherosclerosis have not yet been clarified. A recent animal study conducted on apolipoprotein-deficient mice that were fed a high cholesterol diet showed that administration of CST to the same mice significantly reduced aortic atherosclerotic lesions and attenuated macrophages. Furthermore, by in vitro analysis, the authors determined that CST could suppress atherogenesis by inhibiting inflammatory responses in endothelial cells and macrophages13. In addition, the administration of CST to the mouse hindlimb ischemia model resulted in improvement of blood flow and reduction of necrosis38. Given these considerations, we hypothesize that the effects of CST on the inflammatory process in patients with RA could interfere with the process of atherogenesis and CV risks themselves. However, all of these findings should be interpreted with caution and need to be explored in further, large-scale studies.Limited data indicate that high CST levels could reflect advanced CV disease burden, a burden that is significantly increased in RAThis study has several limitations. First, our study was designed as a cross-sectional study, thereby preventing the establishment of causal relationships. Second, it had single-centre patient inclusion with a relatively small sample size. Additionally, due to technical restrictions, we were not able to measure all inflammatory parameters, which could potentially have an impact on some of the results.In conclusion, this study showed that patients with RA have higher serum CST levels than healthy controls. Furthermore, CST was positively correlated with HAQ and DAS28 scores and disease duration. These findings imply that CST possibly plays a role in RA complex pathophysiology, especially in the severity of inflammation and atherogenesis. However, future larger multicentre longitudinal studies are necessary to explain the association between CST and RA.


One dog developed pulmonary edema following HBOT which is a reported adverse event in humans or may have been a manifestation of progression of the dog's underlying disease. No adverse events were noted in cats or other species. In conclusion, HBOT appeared safe across various indications, although oxygen toxicity affecting the CNS was higher than reports in humans. Future prospective, randomized, controlled trials should evaluate specific clinical indications and outcomes.Hyperbaric oxygen therapy (HBOT) is commonly utilized for various human conditions with a low incidence of major adverse effects (0.002–0.035%). Despite growing use in veterinary patients, there remains a paucity of literature describing its use and associated complications. The purpose of this study was to report clinical use of HBOT in small animals and identify the rate of major adverse events at a university teaching hospital. Electronic medical records were searched for small animals receiving HBOT between November 2012 and February 2020. Data extracted from the medical records included signalment, treatment indication, and adverse events. Treatment sessions totaled 2,792 in 542 dogs, 24 cats, and 10 pocket pets and exotics. Common indications included neurologic injuries (50.4%), tissue healing (31.4%), control of oomycete infection (5.5%), neoplasia or post-radiation injury (5.4%), and various miscellaneous conditions (7.4%). Observed minor adverse events included agitation in two dogs and vomiting in three dogs. The most common major adverse event was central nervous system (CNS) oxygen toxicity in 19 dogs. Central nervous system oxygen toxicity, manifesting as focal or generalized seizures, occurred in 0.7% of treatment sessions, with increasing age ( Hyperbaric oxygen therapy (HBOT) is a simple but effective treatment for a variety of medical conditions –3. TreatAlthough HBOT is an accepted medical therapy in human medicine with research substantiating its efficacy, use in veterinary medicine is comparatively novel with conflicting results –9. FurthThe objectives of this study were to evaluate the clinical use and treatment parameters of HBOT in small animals at a university teaching hospital and to report the frequency of adverse events, with particular interest in the frequency and potential risk factors for CNS oxygen toxicity. The study findings will provide foundational knowledge regarding potential veterinary indications and complications of HBOT and guide future prospective, randomized controlled trials to further evaluate its use in veterinary medicine.2), time at pressure], and occurrence of adverse events. Adverse events were characterized as minor if the HBOT treatment session could be safely continued and no additional monitoring or care was required following the event. Adverse events were characterized as major if the HBOT treatment session was discontinued and alterations were made to subsequent HBOT sessions or additional supportive care was required following the event. Both focal and generalized seizures which occurred during an HBOT session were suspected to be due to CNS oxygen toxicity. A focal seizure was defined as spastic and repetitive movement of the patient localized to a portion of the body during which the patient continued to be responsive to the HBOT operator, but which did not stop upon a distraction . A generalized seizure was defined as spastic movement affecting the entire body during which the patient was unresponsive to distraction, and which was followed by a post ictal period during which the patient demonstrated an abnormal depressed mentation. For both focal and generalized seizures, these were attributed to CNS oxygen toxicity if the signs improved upon reduction in chamber pressure. Patients were not excluded from receiving HBOT if they had a history of prior seizures at the discretion of the clinician overseeing treatment.The electronic medical records of all small animal patients receiving HBOT were collected from a university teaching hospital over the period from November 2012 to February 2020. All hyperbaric sessions were performed in a monoplace chamber with three patient-viewing windows and three-view, live-feed video , with sessions monitored at all times by a veterinarian or veterinary technician certified in chamber use. Chamber certification was provided for each chamber operator by the chamber manufacturer after completion of an online training program and a series of treatment sessions performed with supervision from an individual experienced in the use of HBOT. Data collected included patient species, breed, age, weight, indication for treatment, total number of sessions, details of session parameters [maximum pressure, oxygen flow, fraction of inspired oxygen , total number of treatment sessions, and species (dog/cat). Statistical analysis was performed in Microsoft Excel 2016 using the Data Analysis ToolPak.For statistical analysis, the mean, standard deviation, and range are provided for descriptive data. Further statistical analysis was not performed on special species due to limited data points. The remaining data were assessed for normality, and a Kruskal-Wallis test performed on non-parametric group comparison data with a n = 542, 94.1%) with few cats  and other species . Four dogs received HBOT for two clinically distinct indications within the study period.During the retrospective study period, 2,792 HBOT sessions were performed; patients were primarily dogs (p = 0.40). Data demonstrating number of dogs and sessions for different indications are available in p < 0.001). Protocols used during chamber sessions varied with pressures reported up to 3 atmospheres absolute (ATA). A majority (88.5%) were performed at 2 ATA; pressure was gradually increased to the desired therapeutic level over approximately 15 min. Time at pressure ranged from 30 to 75 min with a majority of treatments spending 45 min at pressure. The chamber was then decompressed gradually over approximately 15 min to 1 ATA (sea level) prior to patient removal.Dogs in the study accounted for 2,632 (94.1%) HBOT sessions. Mean age of dogs was 6.7 ± 3.8 years (range 0.2–16), weighing 18.3 ± 13.2 kg (range 1–71). Dogs received on average 4.9 ± 8.3 sessions (range 1–93). There was no difference in number of sessions between species (p = 0.04). Mean weight was 4.9 ± 2.2 kg (range 1.4–10.1). Cats received on average 5.2 ± 6.0 sessions (range 1–26). The most common treatment indication was for wounds or tissue healing , followed by thrombus or vascular events , urinary obstruction , and intervertebral disc disease .Cats accounted for 124 (4.2%) HBOT sessions. The mean age of cats was 8.8 ± 5.4 years (range 0.4–17), which was significantly greater than that for dogs , with one patient each receiving HBOT for myelopathy, gastrointestinal perforation, and concurrent pneumonia and myositis.Other species consisted of a variety of pocket pets and exotics including two macaws and one each of a capuchin monkey, opossum, bat, gray squirrel, skunk, iguana, and goat accounting for 36 treatment sessions, and an average of 3.6 ± 2.6 sessions per patient (range 1–10). The most common indication for HBOT for these patients was for external wounds or abscesses (n = 2) and vomiting (n = 3). No adverse events were noted during sessions with cats or other species.Minor reported side effects were rare in dogs and were limited to perceived agitation  and generalized seizures . Central nervous system oxygen toxicity occurred during the first session in 6/19 dogs while the remaining 13 dogs experienced CNS oxygen toxicity only after multiple uneventful treatment sessions (session 3–14). Duration of treatment at target chamber pressure at the time of onset of signs of CNS oxygen toxicity was variable and was recorded for 16/19 cases. Two patients experienced signs during initial pressurization and 1 dog after 16 min at pressure. The remaining 13 patients displayed onset of signs between 25 min at pressure and the end of treatment . Atmospheric pressure was recorded in the patient's record in 18/19 cases; 14 cases were at 2 ATA, with two dogs showing signs of focal seizures during pressurization beginning around 1.1–1.5 ATA, and one dog each at 2.2 and 2.7 ATA. In all cases of suspected CNS oxygen toxicity, the patient spontaneously recovered upon return to normal pressure and no long-term effects were reported. In 9 of the 19 cases, additional treatment sessions were performed at a lesser atmospheric pressure than that at which the seizures occurred, with no further adverse events reported. Univariable analysis identified gender and age for inclusion in the multivariate regression model (p = 0.01). Female dogs were 2.4 times more likely to have signs of CNS oxygen toxicity compared to males (p = 0.01) (The most common major adverse effect noted during HBOT sessions was attributed to CNS oxygen toxicity. Signs of CNS oxygen toxicity occurred in 19 dogs (3.5% of dogs) with an incidence rate of 0.7% accounting for all treatment sessions . These son model . Increas = 0.01) .A single dog developed pulmonary edema following HBOT; this may have been a manifestation of pulmonary oxygen toxicity to the lungs or may have been a natural sequelae of the patient's disease process or iatrogenic fluid overload. This dog had been diagnosed with diffuse discospondylitis and aspiration pneumonia a week prior to treatment, and a single HBOT session was elected following surgery for pyometra. The dog developed worsening respiratory signs later in the day after receiving HBOT, and thoracic radiographs were concerning for the development of pulmonary edema. The dog received a single dose of furosemide, intravenous fluids were discontinued, and the respiratory signs resolved.With 2,792 treatment sessions reported here, this is to date the largest collection of data regarding the use of HBOT for a variety of clinical indications across several veterinary species. Common indications included neurologic injury, especially acute myelopathies, wound or tissue healing, oomycete infection control, neoplasia, and a number of miscellaneous conditions based on common clinical indications in people or evidence from research models supporting its use.2, temperature or humidity regulation, or maximal pressurization capacities with unknown effects on treatment outcomes. Within the population reported in this study, treatment parameters varied, although most treatments were performed at 2 ATA for 45 min at pressure, which is comparable to other studies  which can alter membrane lipid peroxidation and enzymatic inhibition in the brain , 19. Reg2 was monitored by the attending staff during all treatments using a portable monitoring system with a sensor placed in the chamber. Elevated CO2 levels were combated with increasing O2 flow to the chamber; anecdotally, partial pressures may temporarily reach 3,000–4,000 ppm during treatments with large, heavily panting dogs. Given that dog size was not a factor associated with increased risk of CNS oxygen toxicity, we suspect that elevated CO2 does not play a role in this adverse effect of HBOT.Elevated partial pressures of carbon dioxide have also been implicated as a factor associated with CNS oxygen toxicity . Carbon In human medicine, medications lowering the seizure threshold have been suspected to contribute to CNS oxygen toxicity, although a definitive association between the use of these medications and seizures during HBOT has not been found. Commonly implicated medications include tricyclic antidepressants and selective serotonin reuptake inhibitors, antibiotics such as cephalosporins, tramadol, and narcotics , 23, 24.It is unclear why age was a risk factor for CNS oxygen toxicity, although this is consistent with findings in people . It is hThe CNS oxygen toxicity rate identified in this population of dogs was higher than that typically identified in humans. This may be due to the higher metabolic rate in dogs compared to people. It is also interesting to note that no signs of CNS oxygen toxicity were identified in cats in this study. A previous study identified minor adverse events in cats including head shaking and lip smacking, but similarly identified no major complications in felines . The lacSeveral species other than dogs and cats were represented in this study, with wound healing as the primary indication for utilization of HBOT and no adverse events seen. While the retrospective data did not provide information sufficient to determine the clinical benefit of HBOT, it appeared safe and could be considered as a treatment option for pocket pets and exotics.A major limitation of this study was the retrospective nature and inherent barriers to data extraction from a patient medical record. While major adverse events were clearly noted in records, it is likely that minor adverse events  were underreported, especially as they may have occurred following the conclusion of the treatment session and their occurrence not associated with recent hyperbaric therapy.The collected data provide information on the variety of conditions for which HBOT was utilized in a veterinary university teaching hospital. Adverse events were rare, with signs of CNS oxygen toxicity occurring most commonly. The signs of CNS oxygen toxicity were limited to a very small population of patients, were self-limiting, and did not recur in subsequent hyperbaric therapy sessions. Thus, this study suggests that HBOT is safe for use across a variety of clinical indications, in various species, and with repeated treatment sessions. However, increasing age was identified as a risk factor for CNS oxygen toxicity, and females were 2.4 times more likely to have CNS oxygen toxicity. Further prospective, randomized, controlled trials should evaluate specific clinical indications and outcomes, to determine those patients that receive the most benefit from HBOT, as well as the ideal protocol for chamber use with attention to potential risk factors to CNS oxygen toxicity and other adverse events.https://doi.org/10.6084/m9.figshare.13244507.v1.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Figshare Data Repository - CM, CK, LE and JS contributed in formulation of the study idea. CM and CK collected data. CM and JS performed statistical analysis and drafted the manuscript. All authors contributed to the manuscript preparation and revision.CK was employed by company Whole Animal Gym. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This was in accordance with the SEM descriptive images. The findings indicate that pre-fluoridated enamel areas were less affected by the acid and showed similar features to the untouched enamel. Moreover, a protective effect of the fluoride treatment against irreversible enamel damage was noted as the surface loss (step-height) was significantly reduced (p = 0.03). The study showed that 3DST analysis is a valuable methodology for detecting and quantifying subtle differences between the surfaces. When exploring the combination of all surface texture parameters, it was revealed that the pre-fluoridated eroded enamel surfaces showed considerable similarity to the untouched enamel.Enamel erosion has become a common clinical finding that often impairs dental esthetics and function. In the current study, we aimed to implement the three-dimensional surface texture analysis (3DST) method to explore the protective effect of fluoride on surface texture prior to erosive conditions. For each of the 50 teeth used in this study, the polished buccal enamel surface was divided into three separate areas: the first area was untouched polished enamel, the two other surfaces were immersed in 0.3% citric acid for 30 s. One was treated with high-concentration  sodium fluoride (NaF) solution prior to acid attack, and the other had no treatment prior to acid exposure. Enamel surface texture and step height measurements were obtained using a high-resolution disk scanning confocal microscope, and SEM images were also acquired. Surfaces treated with fluoride showed fewer variations in 3-D surface texture parameters than the eroded surface compared to the control group ( Dental erosion is associated with extrinsic or intrinsic acid that is not produced by bacteria. Intrinsic factors that may cause dental erosion include anorexia nervosa, bulimia nervosa, and conditions with frequent regurgitation of gastric acids, while extrinsic factors are mostly related with diet, environmental conditions, medications, and lifestyle . In addiSince enamel is a non-vital tissue and is not renewable, early prevention should be a priority before restorative management is considered. Preventive measures that address dental erosion can include dietary advice, stimulation of salivary flow rate, use of buffering medications, and optimization of fluoride regime ,4. FluorQuantitative methods commonly used to describe enamel roughness include profilometry ,7,8,9, eThe usage of various 3-D technologies  can provide an accurate and measurable characterization of surface texture ,16. Dentz-axis, spatial parameters quantify the lateral directionality of the surface, hybrid parameters are a combination of texture amplitude and spacing measurements to characterize the overall nature of surface roughness, functional (volume) parameters indicate material void volume, and feature parameters characterize the projecting surface features  [Another advantage of 3DST is that it enables highly accurate 3-D measurement in irregular surfaces  . The acqd hills) ,18.Therefore, the 3DST methodology can provide a new approach for exploring various experimental and clinical conditions. In the current study, we aimed to implement the 3DST methodology to explore the protective effect of fluoride on enamel surface texture prior to erosive conditions.Fifty fully developed human permanent first and second lower molars were used in this study. All teeth were freshly extracted for periodontal reasons from male and female patients (age range 30–65 years). The study was approved by the ethics committee of Tel Aviv University (Authorization number: 0001117-1). Teeth with caries, restorations, or severe damage were excluded from the study. Once the tooth was extracted, the debris and soft tissue remnants were removed from the root with a sharp scalpel, then it was examined under proper light and magnification to detect any enamel anomalies, such as surface cracks, discoloration, or incipient caries. The teeth were repeatedly washed with double deionized water and then stored in 0.1% thymol solution . Original enamel surface shows great variability in its topography , thus maFor each of the 50 teeth used in this study, the polished buccal enamel surface was divided into three separate areas by placing a borderline made of polyvinyl siloxane material . The first area was untouched polished enamel , the second was treated with highly concentrated  sodium fluoride solution for two minutes prior to acid attack (fluoridated eroded enamel—FEE), and the third had no fluoride treatment and was exposed to acid (eroded enamel—EE). Thorough rinsing and blot drying of the specimens was performed and the FEE and EE surfaces were immersed in 0.3% citric acid, pH 3.2 (adjusted using sodium hydroxide buffer ) for 30 The fluoride solution was prepared by mixing 99% pure sodium fluoride  with double deionized water (pH 7.0) (21 g NaF/500 mL deionized water). Ion-selective fluoride electrode  was utilized to calculate and verify consistency across prepared solutions, after being calibrated before each measurement. All the specimens were left to dry overnight before data collection to ensure consistent scanning of all surfaces under the same scanning condition.Enamel surface texture was imaged using a high-resolution disk scanning confocal measuring system  equipped with a 160L lens (100× magnification) under a total magnification of 1000×. Surface images and texture data were collected using a spatial resolution of x, y = 0.31 µm and axial resolution of z = 0.003 µm. Prior to the measurement, all specimens were visually inspected under low magnification (100×) to identify the borders of the three areas of interest using the same measuring system. Consequently, high-resolution scans of 160 × 160 µm area each were conducted: polished normal enamel (NE), eroded area (EE), and fluoridated eroded area (FEE). Measurements were disregarded if surface area defects were found or if less than 98% of surface points were captured, wherein scans were repeated at a slightly different location. Surfaces were analyzed for several parameters  using µsoft analysis premium software , and filtering operators were employed following ISO/FDIS 25178-2 . For the evaluation of irreversible erosion of dental tissue, the step-height technique  was emplp = 0.05. The outliers were removed from the analysis using the ROUT method (Q = 1%) [Statistical analysis of data from the 50 teeth was performed using GraphPad Prism software , with the level of significance set to (Q = 1%) . The var(Q = 1%) . Kolmogop < 0.001). Maximum peak height (Sp) showed almost equal values for FEE and EE, both higher than NE (p < 0.001). The extreme peak height (Sxp) showed the highest value for the EE group, then FEE, and lowest for NE (p < 0.001). The NE and EE groups showed a negative skewness, while the FEE group had a positive value. Based on the surface texture parameters as defined by the International Organization for Standardization (ISO/FDIS 25178-2), a clear distinction could be noted between the examined areas of the 50 teeth . The resp < 0.001). Moreover, measuring parts of the surface including 5 or 10 voids or hills (five-point peak height five-point (S5p), five-point valley height arithmetic (S5v), 10-point height (S10z)) also showed similar results to the volume parameters for all three groups (p < 0.001). The dale area (Sda), hill area (Sha), as well as both the dale volume (Sdv) and hill volume (Shv), were found to be highest in the FEE group (p < 0.02).The related volume parameters describing the voids , material volume of the core at a given material ratio (Vmc), void volume of the core (Vvc), void volume of the valley at a given material ratio (Vvv)) indicated the smallest voids for the NE group, the largest for EE, and intermediate values for the FEE group  was performed for the 30 surface texture parameters , to present multivariate results on a two-dimensional plot. The graph represents the overall differences between the groups, taking into account the contribution of all 30 parameters, which otherwise would not be feasible to view on a single chart. Therefore, the considerable overlap between the NE and FEE points indicates the similarity of the measured surfaces, thus implying the protective effect of the fluoridation on the enamel surface.The plot  shows a p = 0.03) lower irreversible enamel loss due to acid attack (0.39 ± 0.04 µm) compared to the control area (0.76 ± 0.05 µm) (The discontinuity in surface representing the step height between NE and EE was distinguishably greater compared to the step between the NE and FEE . The flu0.05 µm) .Descriptive topographic images obtained by confocal microscopy imaging  and SEM images (eight teeth) were captured. This was done in order to examine the qualitative characteristics of the tested surface areas . Surface flatness is represented by a height heat map A,D,G. HoExploring the confocal photo simulation images  and fluorapatite [The interaction of fluoride with the enamel can occur in several mechanisms depending on the concentration, agent used, time of application, and the pH of the environment . It was rapatite ,30. Many factors can impact the protective effect of fluoride-containing agents on dental erosion, such as excipients  and abrasives  ,32,33. IIt has been shown in a previous study that, under erosive challenges, two highly concentrated fluoride solutions  afforded significant protection to enamel from erosion compared to a low concentration solution [The methodological parameters chosen for this study were based on the protocol suggested by Austin et al. , who shoThe citric acid used in this study is commonly found in fruit juice drinks and most carbonated beverages. Reports suggest that children start consuming soft drinks at an increasingly younger age with higher intake frequencies . Hence, Given the high sensitivity of the method used in this study, we were able to detect changes in the surface roughness and material loss using step height measurement after 30 s of acid exposure. It was previously shown that changes in surface texture can be noted as early as 10 s after exposure, yet step height changes were noted only 60 s after acidic exposure . The sigThe irreversibility of the material loss and the potential for aggravating this damage through abrasional interactions highlight the need for additional preventive measures. Moreover, in cases susceptible to high erosion conditions due to gastroesophageal reflux disease, eating disorders, or exaggerated dietary acid consumption ,33, addiThe values found in our study concur with previous observations that used the arithmetic average roughness parameter (Ra) ,8,37,38,The protective effect of fluoride in maintaining the integrity of enamel from chemical breakdown in acidic environments is significant in the SEM images C,F,I andThe advantage of 3DST is that it reveals the pattern of the surface modification. The height parameters analyzed in this study  exhibited significant differences between the three groups, indicating their potential for diagnosing the erosional and protective changes of the dental enamel surface . We founThe same pattern was also noticed in the volume parameters , where the FEE group showed significantly lower values compared to the EE group. These findings clearly demonstrate the rationale behind etching enamel surfaces in restorative dentistry treatment to increase retention as larger core voids are correlated with increased micromechanical retention. Additionally, it explains the opposite rationale of not fluoridating the enamel surface prior to bonding procedures  .Common surface roughness measurements usually include the Sa  or Ra . It expresses, as an absolute value, the difference in the height of each measured point compared to the arithmetical mean. This means that in case of a flat surface with a large void, for instance, the surface will present a high Sa value although most of it is flat. This measurement disregards the volume of the void and the diameter of the void as it only considers the height parameters. Considering only one value to explore the surface pattern cannot fully represent the texture of the surface.In our study, the Sa parameter showed significant differences between the three groups ; however, when exploring the combination of all features , it was The study showed that 3DST analysis is a valuable methodology as it allows detection and quantification of subtle differences between the surfaces. When exploring the combination of all surface texture parameters it was revealed that the pre-fluoridated eroded enamel surfaces showed considerable similarity to the untouched enamel.


Axenfeld–Rieger syndrome (ARS) is a rare autosomal dominant hereditary disease characterized primarily by maldevelopment of the anterior segment of both eyes, accompanied by developmental glaucoma, and other congenital anomalies. FOXC1 and PITX2 genes play important roles in the development of ARS.The present report describes a 7-year-old boy with iris dysplasia, displaced pupils, and congenital glaucoma in both eyes. The patient presented with a congenital atrial septal defect and sublingual cyst. The patient’s family has no clinical manifestations. Next generation sequencing identified a pathogenic heterozygous missense variant in FOXC1 gene  in the patient. Sanger sequencing confirmed this result, and this mutation was not detected in the other three family members.To the best of our knowledge, the results of our study reveal a novel mutation in the FOXC1 gene associated with ARS. Axenfeld–Rieger syndrome  is an autosomal dominant hereditary disorder, involving malformation of the anterior segment of the eye during development and causing systemic malformations . The incTwo major developmental transcription factor genes, forkhead box C1 (FOXC1) on chromosome 6p25 and pituitary homeobox 2 (PITX2) on chromosome 4q25, have been demonstrated to cause the ARS disease, accounting for 40 to 70% of cases [The patient was a 7-year-old boy in the southwest of China, who had blue cornea, photophobia, and poor vision from birth. When the subject was 36 days old, he had binocular anti-glaucoma surgery, which was followed by long-term treatment with travoprost and brinzolamide eye drops. However, the intraocular pressure (IOP) was not well controlled. The patient underwent surgeries to repair an atrial septal defect causing congenital heart disease and resection of a sublingual cyst. Recently, the eye symptoms worsened and the patient’s vision declined significantly. Thus, the boy and his family came to the Affiliated Hospital of Southwest Medical University. A three-generational pedigree of the patient’s family is shown in Fig. Ophthalmic examination of the patient revealed similar pathological changes in both eyes. Uncorrectable poor vision  and horizontal tremors were observed. No hyperemia or edema was detected in the conjunctiva. The cornea was transparent but the sclera was pale blue. The diameter of the cornea was 14 mm and the center of the anterior chamber was deep. The aqueous humor was clear. The texture of the iris was unclear with partial depigmentation. The lamellar and incomplete disconnections of the root of the iris formed some iridocyridosis and slit-like iris holes  Fig. A, B. TheAccording to the clinical manifestations, the patient was diagnosed with ARS (Type 3), bilateral congenital glaucoma, and bilateral congenital cataracts. To treat the glaucoma and relieve optic nerve atrophy, the patient underwent a trabeculectomy and iris circumferential resection in the right eye. After surgery, the IOP was 15 mmHg in the right eye. The IOP in the left eye dropped to 16 mmHg after treatment with anti-glaucoma drugs.Considering the possibility of congenital disease, three of the boy’s family members  underwent complete ophthalmologic and systemic examinations, but no positive findings were observed. Genetic tests were performed on the patient.This study was performed in accordance with the tenets of the Declaration of Helsinki. Approval to conduct this study was obtained from the Ethics Committee of Affiliated Hospital of Southwest Medical University (KY2019110). Informed consent was obtained from the guardian (parents), who agreed to join this study, and for the use of medical information and images for scientific research and publication.With consent, all family members  had genetic testing. Peripheral blood from each family member was collected and sent to MyGenostics for testing. DNA samples from the patient were genetically tested using an NGS-based gene panel. For exome sequencing, we fragmented 1–3 μg of genomic DNA to an average size of 180 bp with a Bioruptor sonicator (Diagenode). Paired-end sequencing libraries were prepared using a DNA sample prep reagent kit (NEBNext). Library preparation included end repair, adapter ligation, and polymerase chain reaction enrichment. These procedures were performed following Illumina protocols. Amplified DNA was captured using a Gencap Capture kit (MyGenostics GenCap Enrichment technologies) containing probes for 50 genes related to iris disease. DNA probes were designed to tile along the exon regions of genes related to iris diseases. The enrichment libraries were sequenced on Illumina Nova 6000 sequencer for a paired read of 150 bp . After sequencing, the raw data were saved as a FASTQ format and bioinformatics analyses were performed.After removing the low-quality reads (< 80 bp), contamination and linkers from the raw data, the clean reads were mapped to the UCSC hg19 human reference genome using BWA software. The single nucleotide variations (SNVs) as wells as inserts and deletions (INDEL) were identified using GATK software, followed by annotation using ANNOVAR software. The mutations with minor allele frequencies (MAF) less than 0.02 were excluded, which included the 1000 Genomes Project, ESP6500, EXAC and gnomAD databases. The pathogenicity and conservatism of missense mutations were predicted using SIFT, PolyPhen-2, MutationTaster, GERP++ and REVEL. The pathogenicity of splice site changes was analyzed using SPIDEX. Finally, four suspected pathogenic variants were identified. Among them, three recessive heterozygous variants, CEP290 , ELP4 , and OCA2 , were not associated with the present clinical phenotype and were excluded  in FOXC1 in a patient with ARS type 3, and our report may be useful for better understanding of the spectrum of FOXC1 mutations and provides further information about the phenotypic characteristics of ARS patients. Furthermore, the hybrid recessive mutations of CEP290, ELP4, and OCA2 were also detected in the proband, but may have nothing to do with the clinical phenotype. However, further study is needed.