In silico analyses of bacterial mutagenicity have recently gained acceptance by regulatory agencies; however, current in silico models for prediction remain to be improved. The Japan Pharmaceutical Manufacturers Association (JPMA) organized a task force in 2017 in which eight Japanese pharmaceutical companies had participated. The purpose of this task force was to disclose a piece of pharmaceutical companies’ proprietary Ames test data.Ames test is used worldwide for detecting the bacterial mutagenicity of chemicalsuvrA, or their combination.Ames test data for 99 chemicals of various chemical classes were collected for disclosure in this study. These chemicals are related to the manufacturing process of pharmaceutical drugs, including reagents, synthetic intermediates, and drug substances. The structure-activity (mutagenicity) relationships are discussed in relation to structural alerts for each chemical class. In addition, in silico analyses of these chemicals were conducted using a knowledge-based model of Derek Nexus (Derek) and a statistics-based model (GT1_BMUT module) of CASE Ultra. To calculate the effectiveness of these models, 89 chemicals for Derek and 54 chemicals for CASE Ultra were selected; major exclusions were the salt form of four chemicals that were tested both in the salt and free forms for both models, and 35 chemicals called “known” positives or negatives for CASE Ultra. For Derek, the sensitivity, specificity, and accuracy were 65% (15/23), 71% (47/66), and 70% (62/89), respectively. The sensitivity, specificity, and accuracy were 50% (6/12), 60% (25/42), and 57% (31/54) for CASE Ultra, respectively. The ratio of overall disagreement between the CASE Ultra “known” positives/negatives and the actual test results was 11% (4/35). In this study, 19 out of 28 mutagens (68%) were detected with TA100 and/or TA98, and 9 out of 28 mutagens (32%) were detected with either TA1535, TA1537, WP2The Ames test data presented here will help avoid duplicated Ames testing in some cases, support duplicate testing in other cases, improve in silico models, and enhance our understanding of the mechanisms of mutagenesis.The online version contains supplementary material available at 10.1186/s41021-021-00206-1. The four pairs of these chemicals showed the same (negative) result with a similar toxicity between each pair, except for a pair of chemical IDs 28 and 29. Individual data are shown in Although some chemical classes have only a few chemicals, we discuss the structure-activity (mutagenicity) relationships in relation to structural alerts.N-hydroxylamines by bacterial nitroreductase, and therefore can be efficiently detected in the absence of S9 mix [The structure of nitroarenes is a representative alert for mutagenicity, although the simplest nitroarene nitrobenzene itself is not mutagenic –16. All f S9 mix –16. Intef S9 mix , 16.N-hydroxylations, typically by the CYP 1A2 enzyme, followed by O-esterification with acetate or sulfate [N-hydroxylamines and form adducts with DNA, leading to mutations [para-iminoquinone, which does not require metabolic enzymes.The structure of aromatic amines is also a representative indicator of mutagenicity –14. The  sulfate , 18. In utations . The mutThe 2-aminothiazoles tested, which were five-membered aromatic amines containing hetero atoms of sulfur in position 1 and nitrogen in position 3, were half mutagenic  and half non-mutagenic , with a diverse substituent at position 4. 2-Aminothiazoles were all predicted to be mutagenic (as “Plausible” by Derek) through identification of the structural alerts of aromatic amines or amides. 2-Aminothiazole is mutagenic, and the mutagenicity of 2-aminothiazoles is induced via the formation of reactive nitrenium ion intermediates, such as aromatic amines –21. The The six quinolinone derivatives  were non-mutagenic, whereas the other three were mutagenic. The quinolinone structure was not an alert, as shown by both in silico models. Chemical ID 19 was mutagenic, probably because of the presence of epoxide. The mutagenicity of chemical ID 20 may be derived from the dihydroxylated piperazine moiety. Chemical ID 21, an 8-hydroxy derivative of quinolinone, was mutagenic only in TA1535, and TA1537, which shows a small number of negative control counts and is empirically known to be sensitive to some structures.uvrA, WP2uvrA/pKM101, or TA102, which have an AT base pair at the primary reversion site [uvrA/pKM101 [uvrA was used, the three fluoroquinolone derivatives, including grepafloxacin  and grepafloxacin HCl , were all non-mutagenic.The mutagenicity of fluoroquinolones was dependent on WP2ion site . Fluoroqion site  and WP2uA/pKM101 , and theThe difference of cytotoxicity  in the two forms  was much more than would be expected by normal variation. It may be worth looking at the role of the different solvents, including water and DMSO.The five pyrimidinedione derivatives were all non-mutagenic. Both in silico models predicted these chemicals to be inactive/negative except for one chemical called the “Out of Domain” owing to the presence of two trimethylsilyl moieties, as shown by CASE Ultra. The structure of pyrimidinedione should not be an alert for mutagenicity.All three triazole derivatives were non-mutagenic. Both in silico models predicted that these chemicals were inactive/negative except for the “Inactive containing unclassified features” and “Out of Domain” owing to the presence of a tertiary amine moiety, as shown by Derek and CASE Ultra, respectively. The structure of triazole is unlikely to be an indicator of mutagenicity.The two heterocyclic compounds, derivatives of oxadiazole  and 1,3,5-triazine , were both non-mutagenic. The finding that chemical ID 39 was non-mutagenic was not consistent with the “known positive” from CASE Ultra.The three sulfonyl derivatives were all non-mutagenic, which was consistent with that in both in silico models, although Derek identified an unclassified feature of sulfonimide in chemical ID 41. The structure of the sulfonyl moiety is not an alert for mutagenicity.Chemical IDs 44 and 45 were both mutagenic, and this result was consistent with the results of both in silico models. Several sulfonate esters are well-known to be alkylating mutagens, and predicted as “plausible” mutagens by Derek. However, chemical ID 46 was not mutagenic. The mutagenic potency of sulfonates is dependent on both the leaving group and alkylsulfonate moiety, affecting their chemical reaction rate , 25 and The two sulfonyl chlorides  and benzoyl chloride  were mutagenic in the presence or absence of S9 mix. Dimethyl sulfoxide (DMSO) was used as the solvent. It was reported that when DMSO was used to dissolve sulfonyl chlorides or acyl chlorides (including benzoyl chlorides), these chemicals showed mutagenicity  due to the generation of mutagenic impurity (chlorodimethyl sulfide) in the test chemical formulations, with a few exceptions , 30. Der2 reaction through steric hindrance by the bulky substituent around the carbon center adjacent to the chlorine atom. In this study, chemical ID 54 with a long alkyl chain (hexyl moiety) and a leaving group of bromine ions is marginally positive only in TA1535, which shows a low number of negative control counts in the presence of S9 mix, although n-butyl chloride with a shorter alkyl moiety is reported to be non-mutagenic [Halogenated alkanes  can be alkylating mutagens without requiring metabolic activation. Similar to that of sulfonate esters, their mutagenic activity is dependent on the alkyl moieties and the leaving group of halogen ions. A possible reason why chemical IDs 55 and 56 were non-mutagenic is that the DNA adduct was not formed via inhibition of the SNutagenic . PrimaryuvrA; the strains TA100 and TA98 were most sensitive among the five strains that are recommended for use by OECD test guideline 471 [The two halogenated benzenes were non-mutagenic. Chemical ID 57 was tested with three test strains, TA100, TA98, and WP2line 471 . HalogenFive 4,6-dibromo-3-fluoro-2-methylbenzoate derivatives  were non-mutagenic, and Derek and CASE Ultra did not show alerts for this structure. Therefore, the structure of 4,6-dibromo-3-fluoro-2-methylbenzoate is not an indicator of mutagenicity. The mutagenicity of chemical ID 59 might involve the enol (tautomerized) form of the 1,3-diketone moiety, followed by epoxidation of the double bond by the drug-metabolizing enzyme in S9 mix. The substitution at position 2 of the 1,3-diketone moiety may inhibit tautomerization, but not lead to the induction of mutagenicity . It remains unclear why chemical ID 60 was mutagenic. Mutagenicity may be associated with the magnesium-oxygen complex.Both cinnamyl esters were non-mutagenic, as predicted by both in silico models. A double bond conjugated with a benzene ring is unlikely to be a structural indicator of mutagenicity.All benzoates were non-mutagenic, as predicted by both in silico models.Phosphorus-containing chemicals were all non-mutagenic except for chemical ID 71, which is electrophilic and routinely used in organic synthesis for the phosphorylation of amines . For manCyanide ion  and all the cyanide derivatives substituted with an aromatic ring were non-mutagenic. The cyanide moiety is unlikely to be a structural alert for mutagenicity, as supported by Derek.Chemical ID 81, an aldehyde conjugated with a single carbonyl moiety, was mutagenic, as predicted by both in silico models. The chemical properties of aldehydes largely differ between aliphatic and aromatic compounds; generally, the former is chemically reactive, whereas the latter is stable. Both aromatic aldehydes  were non-mutagenic, which can be explained by the extremely low chemical reactivity of aromatic aldehydes.uvrA and TA1535, respectively. Both chemicals were predicted to be mutagenic  by both in silico models. Chemical ID 87 was only mutagenic in TA1537, which would be a tester strain sensitive to some chemical structures, with a small number of negative control counts.The miscellaneous group consists of chemicals that cannot be simply classified into the above chemical classes. Many of the chemicals tested were non-mutagenic. Chemical ID 84 was mutagenic in the presence and absence of S9 mix, although there were no structural alerts identified by Derek. The cause of the mutagenicity is unclear, but aldehyde might be involved in the induction of mutagenicity, which may be generated from alcohol by the alcohol dehydrogenase present in bacteria . The thruvrA). For chemical IDs 60 and 99, the in silico models could not reach a conclusion because the former is a complex molecule and the latter is a radical. We treated “Out of Domain” fragments as well as “Inconclusive”, “Equivocal”, “Inactive (contains misclassified or unclassified features)”, as neither Ames-positive nor Ames-negative in this study.To calculate the sensitivity, specificity, and accuracy of in silico predictions, ten chemicals  were excluded. Four chemicals tested in both forms were used for calculation in the free form , but not in the salt form . Chemical IDs 47–49 were false positive because probable inappropriate solvents were used. Chemical ID 57 was tested in only three strains  revealed the sensitivity, specificity, and accuracy to be 65% (15/23), 71% (47/66), and 70% (62/89), respectively. In contrast, in silico analysis using CASE Ultra  revealed the sensitivity, specificity, and accuracy to be 50% (6/12), 60% (25/42), and 57% (31/54), respectively. Thus, Derek outperformed CASE Ultra (GT1_BMUT) in the predictive level of bacterial mutagenicity for all the parameters in this study, where the limited number of chemicals were compared.Derek and Case Ultra occasionally called “inactive containing misclassified or unclassified features” , and “Out of Domain” fragments , respectively, indicating the need to expand the training or reference set for each in silico model to improve.It is worth noting that when considering the performance of the in silico models, it is important to account for the ICH M7 approach of combining two complementary systems and an expert review to take a final decision rather than considering them separately , 34., plate-incorporation method vs. preincubation method, the type of strains used, source of test strains, preparation of overnight culture), and evaluation criteria , and quality of test substances [The 35 chemicals (15 “known” positives and 20 “known” negatives) were part of the training set for CASE Ultra. The results for 4 of 35 chemicals 11%) did not agree with the known response for those chemicals in that training set. The four chemicals  were non-mutagenic but were registered as mutagens in the training set for CASE Ultra. This disagreement ratio (11%) was in the same range as the Ames test non-reproducibility, identified by Piegorsch and Zeiger, who reported a value of approximately 13% [bstances , 11, 36.% did notTwo chemicals  were mutagenic but were registered as non-mutagens in the CASE Ultra training set. This is probably because the solvent used in our study was not appropriate, as previously stated  were mutagenic. Among them, three chemicals , two chemicals , two chemicals , and two chemicals , respectively, were only detected for mutagenicity in either TA1535, TA1537, WP2s et al.  reportedAmes test data were presented for 99 chemicals from eight pharmaceutical companies through the activity of the Ames data sharing task force. The chemicals were related to the manufacturing process of pharmaceutical drugs, including reagents, synthetic intermediates, and drug substances. The Ames test data presented herein will contribute to avoiding duplicated Ames test in some cases, supporting duplicate testing in other cases, improving in silico models, and enhancing our understanding of the mechanisms of mutagenesis.Additional file 1.


SLC4A1, RHAG, ANK1, MKNK2, SPTB, ADA, C7 and EPB420 genes were highlighted and should be further explored to validate them as candidates to FHS and FHN in chickens and possibly in humans.Femoral head necrosis (FHN) and other locomotor problems cause severe impacts on the poultry industry due to huge economic losses and reduced animal welfare. Femoral head separation (FHS), the initial phase of FHN, is usually a subclinical condition characterized by the detachment of articular cartilage from the bone. In this study, we aimed to identify genes and biological processes involved with FHS in broilers. A better understanding of the FHS molecular mechanisms can help to develop strategies to reduce this condition in chickens. Here, we described several genes that have their expression altered in the articular cartilage and femur when normal and FHS-affected animals were compared. Furthermore, genetic variants were found differing between the studied groups. Therefore, performing an integrated analysis of these datasets, we were able to detect genes and variants related to FHS in chickens. Some of them, such as SLC4A1, RHAG, ANK1, MKNK2, SPTB, ADA, C7 and EPB420) were shared between chickens and humans. Furthermore, 89 variants, including missense in the SPATS2L, PRKAB1 and TRIM25 genes, were identified between groups. Therefore, those genes should be more explored to validate them as candidates to FHS/FHN in chickens and humans.Femoral head separation (FHS) is usually a subclinical condition characterized by the detachment of articular cartilage from the bone. In this study, a comprehensive analysis identifying shared and exclusive expression profiles, biological processes (BP) and variants related to FHS in the femoral articular cartilage and growth plate in chickens was performed through RNA sequencing analysis. Thirty-six differentially expressed (DE) genes were shared between femoral articular cartilage (AC) and growth plate (GP) tissues. Out of those, 23 genes were enriched in BP related to ion transport, translation factors and immune response. Seventy genes were DE exclusively in the AC and 288 in the GP. Among the BP of AC, the response against bacteria can be highlighted, and for the GP tissue, the processes related to chondrocyte differentiation and cartilage development stand out. When the chicken DE genes were compared to other datasets, eight genes ( The poultry industry has experienced huge growth in recent decades, due to the advances in new research and technologies, which has brought benefits to the industry and producers. Chicken meat is one of the most consumed proteins in the world, due to the low cost, for being considered cost-effective and healthy, and because it is exempted from cultural impediments . The conThe genetic breeding programs have brought numerous benefits for poultry production, such as the increased body weight, greater thoracic musculature, and reduction of abdominal fat. In the last 50 years, chicken growth rates have risen more than 300%, from 25 g per day to approximately 100 g per day . HoweverLocomotor problems cause severe impacts on the poultry industry, affecting about 6% of the animals in commercial flocks, which results in huge economic losses . The bacSince the etiology of FHN remains unclear, studies are being conducted to clarify its pathogenesis and the molecular mechanisms involved, and to discover genes with a potential role in triggering FHS and subsequent BCO in the head of the femur and tibia ,15,16,17Transcriptomes used in the present study were obtained from a project that evaluated femoral head separation (FHS) in the growth plate and articular cartilage, published respectively by Peixoto et al.  and Hul https://github.com/hanielcedraz/BAQCOM, accessed on 31 December 2021) was used for quality control (QC) and mapping. QC for each sequencing file was performed with the Trimmomatic v. 0.38 [https://www.ensembl.org/biomart, accessed on 31 December 2021). Visualization and Integrated Discovery (DAVID 6.8) [http://pantherdb.org/, accessed on 31 December 2021) [The baqcom pipeline ( v. 0.38  to remov v. 0.38  was used v. 0.38 . PositivVID 6.8)  and Panter 2021)  databaseSLC4A1), Rh- associated glycoprotein (RHAG), ankyrin 1 (ANK1), MAP kinase interacting serine/threonine kinase 2 (MKNK2), spectrin beta, erythrocytic (SPTB), adenosine deaminase (ADA), complement C7 (C7) and erythrocyte membrane protein band 4.2 (EPB42). Gene sequences were downloaded from Gallus gallus on Genebank  and Ensembl . Primers were designed in exon–exon junctions using the Primer-Blast online tool [http://www.premierbiosoft.com/netprimer/, accessed on 31 January 2022) , since the RHAG, ANK1 and ADA genes have already been confirmed in the analyzed population by Hul et al. [−ΔCt [p ≤ 0.05.A relative quantification using qPCR was performed to confirm the expression profile of eight chosen DE genes in the GP using eight samples from the four normal and four from the FHS-affected group. The genes selected were solute carrier family 4 member 1  . For AC,l et al. . Ribosoml et al.  and Hul l et al. , respectl. [−ΔCt . StatistTo identify the main molecular mechanisms involved in FHS, the common and exclusive DE genes in the AC and GP datasets were obtained, and the David  databasehttps://www.ncbi.nlm.nih.gov/gds, accessed on 9 November 2020) to search datasets with the terms “femoral head necrosis” and “epiphysiolysis” to better understand the role of the main DE genes involved with FHS across different species. This search returned two main datasets: identification of potential biomarkers for improving the precision of early detection of steroid-induced osteonecrosis of the femoral head (GSE123568) and gene expression profile of hip cartilage with necrosis of femoral head (GSE74089). The first one evaluated the gene expression on blood from 40 patients  and the second one, the hip cartilage in 24 patients . DE genes were obtained using the GEOR tool from the GEO dataset . The list of DE genes from the two datasets obtained in the GEO database and the DE genes found in the GP and AC from our study were submitted to the InteractiVenn  [Furthermore, we used the Gene Expression Omnibus (GEO) database . The Picard tools 2.5  was used to generate the genome index, to assign read groups and mark duplicates. In the GATK, the CIGAR strings determination (SplitNCigarReads), qualities reassigning mapping, base recalibration, variants calling, and filtering were performed. To minimize the false positive variants, the following filters were used in GATK to filter and select variants: FS > 30.0, MQRankSum < −12.5, SNPcluster considering 3 variants in a 35 bp window, QD < 5.0, MQ < 50.0, GQ < 5.0, QUAL ≥ 30.0, ReadPosRankSum < −8.0 and DP ≥ 100.0. Furthermore, variants with less than 10 reads per sample were removed from the downstream analysis. Once the polymorphisms were identified in the GP and AC datasets, a filter was performed to obtain the variants that differed between normal and FHS-affected groups. The Variant Effect Predictor (VEP) tool [Gallus gallus genome (GRCg6a) with the Ensembl 103 annotation database version. Furthermore, the Stringdb [The Genome Analysis Tool kit 3.6 (GATK)  was usedEP) tool  was usedStringdb  and EnriStringdb ,34 databIncRNA, pseudogenes, miRNAs and other noncoding RNAs , and after the quality control, approximately 306 million reads (88.78%) remained for further analyses , with aning RNAs .Using the Ensembl annotation 101, in the AC transcriptome, 12,182 genes were identified and 106 genes were DE; as well, 99 (93.44%) were upregulated and seven (6.56%) were downregulated in the AC of the FHS-affected compared to the normal group . RegardiComparing the DE genes between the AC and the GP, 36 DE genes were common to both transcriptomes . From thThe enrichment analysis performed based on the common DE genes between the two tissues showed that the main biological processes found were related to ion transport, inflammatory and defense response, homeostasis, protein biogenesis and immune response processes .The most relevant BP for the AC tissue was the defense response to bacterium, cell chemotaxis and regulation of cell motility . ConversTF) gene is involved in antimicrobial activity, while some genes such as Hemoglobin subunit gamma-2 (HBG2) and Rh-associated glycoprotein (RHAG) are involved in oxygen and ion transport. Another group connected the PTPRC, CCL10, ISG12-2 and SAMD9L genes that are most related to the immune response BP  were DE. The MKNK2 and SPTB genes were also DE in the AC. Furthermore, all evaluated genes were upregulated in the FHS-affected group , EPB42 (Erythrocyte Membrane Protein Band 4.2), SPTB  and ANK1 (Ankyrin 1)  evinced 5250 DE genes in the blood of those individuals, while 5978 DE genes were found in the hip cartilage of normal and FHN-affected individuals (GSE74089). Considering these four datasets, four genes were DE in all studies: kyrin 1) A, all ofSLC4A1, EPB42, ANK1 and SPTB) and four additional genes: CCL26 (Chemokine (C-C motif) ligand 26), ADA (Adenosine Deaminase), SLC25A37 (Mitoferrin-1) and MKNK2 (MAPK Interacting Serine/Threonine Kinase 2), all involved with immune response  and bone datasets to verify the presence of shared genes in the tissues affected by FHN/FHS. In this comparison, eight shared genes were found: the four previously described  gene, another SNP overlapped the downstream region of two genes, CCM2 Scaffold Protein (CCM2) and NAC Alpha Domain Containing , and the other was a missense variant in the Staphylococcal Nuclease and Tudor Domain Containing 1 . Variants were mainly classified as downstream gene variant, synonymous variant, upstream gene variant, intron variant, 3′ prime UTR variant and missense variants  were found in the analyzed samples with the GATK tool. Out of those, 89 differed between the normal and FHS-affected groups . These 8variants .PRKAB1 (rs733851944), SPATS2L (rs737569658) and TRIM25 (rs314175171), with a Sorting Intolerant from Tolerant (SIFT) score of 0.36 and 0.16. The novel variant (chr1:1049560) in the SND1 had a SIFT = 0, being predicted as deleterious.Out of the described coding variants, 90% was classified in synonymous variants, 10% was missense . ConsideLOXL2 gene and the other in the amino acid position 52 of the SND1 (ENSGALG00000054297) gene.After identifying the variants that differ between the normal and affected groups in the RNA sequences, a search was performed to verify if some of the variants were present in the DE genes in both AC and GP tissues previously found between normal and FHS-affected samples. None of the variants were found in the common DE genes; however, two polymorphisms were found in the DE genes in the GP: a SNP (rs737937191) in the downstream region of the https://www.animalgenome.org/cgi-bin/QTLdb/GG/index, accessed on 31 December 2021), such as a SNP in the RPS24 gene, located in a QTL region associated with tibia volume  (SLC25A3), Yip1 Domain Family Member 3 (YIPF3), DEAD-Box Helicase 55 (DDX55), Exportin 5 (XPO5), RNA Polymerase I And III Subunit C (POLR1C), R3H Domain And Coiled-Coil Containing 1 (R3HCC1), Transmembrane P24 Trafficking Protein 2 (TMED2), Ras Responsive Element Binding Protein 1 (RREB1), charged Multivesicular Body Protein 7 (CHMP7), DNA Polymerase Eta (POLH) and Lysyl oxidase homolog 2 (LOXL2) have already been associated with waist-to-hip ratio adjusted for body mass index (BMI) in humans  . Furthern humans .ADA gene was DE in AC and GP and grouped with several genes, which have SNPs differing between normal and affected groups, such as the TRIM25, LUM, MYH9, RPS24 and NACAD.The interactions between the DE genes and genes with variants were verified through a gene network, where it was possible to observe that those genes have physical or functional interactions and were grouped in five main subnetworks . For exaThe mechanical stress caused by the overload of the skeleton that modern chickens sustain due to the high growth rates has been recognized as one of the main causes of locomotor problems ,12,35. ARUNX2 and SPARC genes may be associated with reduced vascularization and poor bone mineralization, increasing the risk of skeletal problems in chickens. The impaired collagen formation, connective tissue cell adhesion, and bone extracellular matrix (ECM) were also identified as predisposing factors for BCO and other leg disorders [There are few studies related to FHN and other bone integrity problems in chickens, especially those approaching the molecular mechanisms involved with these conditions. Previous studies from our group have shown some biological processes (BP) and genes associated with FHN/BCO in different ages and lines of chickens ,15,16,17isorders ,15,16,17isorders . HoweverIn the global transcriptome characterization of the GP and AC tissues, about 12,000 genes were expressed in both tissues. From those, 11,141 were shared and 891 and 441 genes were exclusively to the GP and AC, respectively. As expected, due to the RNA sequencing methodology used, approximately 95% of the genes were coding, although lncRNAs, miRNAS, mitochondrial and miscRNAs were also identified in the analyzed datasets .A total of 36 DE genes were shared by both tissues, and 91.6% of the common genes were upregulated in the FHS-affected group . Some ofTF, HBG2, ANK1, SLC25A37, EPB42, RHAG, ADD2, KEL, MKNK2 genes and another by the PTPRC, CCLI10, ISG12-2 AND SAMD9L genes. In general, the first group of genes are related to blood type and to cartilage regeneration, and the second is related to inflammatory response [Two main branches were observed in the common DE genes network : one wasresponse ,37. MoreTF) was in the main branch of the gene network and its primary function is to transport iron to tissues that require this mineral [TF has a function in endochondral ossification, being considered the angiogenic molecule released by the hypertrophic cartilage [TF presented strong interactions with the HBG2 (Hemoglobin Subunit Gamma 2) gene, which is involved in the transport of oxygen from the lungs to the peripheral tissues, where the beta chain is a component of adult hemoglobin A and D [HBG2, as the TF, was upregulated in the FHS-affected broilers (HBG1 and HBG2) are expressed in several tissues, such as fetal liver, spleen and bone marrow [The transferrin  works as a solute transporter located in the internal mitochondrial membrane, importing iron, which is essential for the synthesis of mitochondrial heme and iron–sulfur clusters [BMP13, BMP14 and BMP15 were responsible for regulating genes of the SLC family [The clusters . In geneclusters  and alsoclusters . In humaC family .ANK1 gene encodes a binding protein of the cytoskeleton, helping to bind other membrane proteins to the actin–spectrin cytoskeleton [ANK1 is usually found in erythrocytes, but it has already been found in the brain and muscles [ANK1 can possibly affect the actin structure of the cytoskeleton, changing the structural integrity of the femoral articular cartilage and contributing to the occurrence of FHS. In addition, its expression is related to cell damage, which can be a consequence of FHS, because when FHS starts, positive regulation can act as a sign of attempt to combat the progression of this condition. This gene is also involved in the upregulation of inflammatory cytokines in osteoarthritic lesions [ANK1 gene is regulated by the EPB42, another gene that was upregulated in the FHS-affected group. The ANK1, EPB42 and RHAG  gene interacts with MAP kinase and may respond to environmental stress and cytokines. Among its related pathways are the interleukin 1 signaling pathway. MAP/ERK signaling acts as a mediator of the suppressive effects of IFN-gamma in hematopoiesis [MKNK2 is not normally expressed in rats, but appears in stressful situations, showing its adaptive function and can also be a signal for cellular apoptosis [The opoiesis . The MKNpoptosis . BringinMYH15, has also been associated with the adrenergic signaling process in cardiomyocytes, in cases of imbalance in the O2 supply and removal of CO2 from tissues [Another DE gene, the  tissues . This geEPX gene is released during the immune response by eosinophils, having a cytotoxic effect on cells [EPX gene has been studied as a biomarker for inflammatory events in the gastrointestinal tract [EPX upregulation in broilers affected with FHS may indicate the immune system response to inflammation and, in severe cases, may be related to local necrosis.The on cells ,55 and bon cells . The eoson cells ,58. In cal tract  and has al tract ,17. The ADA and RHAG also participate in the process of metabolic glycosaminoglycans (GAGs) and aminoglycans involved in the metabolism of ECM [ADA enzyme acts as an endogenous regulator of the adaptive immune system, especially on proliferation and differentiation of T lymphocytes, regulating cell metabolism and triggering several physiological effects on cell proliferation [The extracellular matrix (ECM) is a stable structural component that is located under the epithelium and close to connective tissue , and is m of ECM , indicatferation ,64.ADA gene also acts as a sensor providing information to the immune system regarding tissue damage, protecting cells from excessive tissue damage associated with inflammation [ADA has a regulatory role in immune responses, acting on the activation and regulation of lymphocyte and neutrophil levels [ADA was upregulated in AC and GP in the FHS-affected group  is a member of a family of collagen-associated small leucine-rich proteins (SLRPs) responsible for signaling of differentially regulated collagen fibrils during development, homeostasis, or pathogenesis. CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis and negatively modulates chondrocyte differentiation [Furthermore, once the exclusively DE genes in the GP were evaluated, the main biological processes were those related to chondrocyte differentiation and cartilage development, grouping three main genes that had the lowest expression in the affected group: nd PTHLH . Chondrontiation .GDF5 gene expression is related to joint tissue remodeling. After joint damage, its high expression in chondrocytes is observed, both in the new cartilage recovery tissue and in the adjacent damaged cartilage [PTHLH gene promotes the proliferation of chondrocytes and inhibits their hypertrophy and terminal differentiation. In patients with osteoarthritis, this gene is highly expressed in chondrocytes and synovial fluid [In humans, increased artilage . The PTHal fluid .CAMP) and cathelicidin-3 (CATH3) stand out, which have a broad spectrum of antimicrobial activity and the ability to limit inflammation by inhibiting the activation of Toll-like receptor 2 (TLR2) and TLR4 [Among the BP of the AC, the response against bacteria can be highlighted, with all genes involved in this BP being more expressed in the FHS-affected group . Among tand TLR4 .These results suggest that the growth plate is not fully efficient in the process of recovery and remodeling of damaged cartilage. Conversely, articular cartilage can partially control bacterial infection and the intensity of inflammation that can intensify tissue damage. Other mechanisms should be evoked.SLC4A1 (Solute Carrier Family 4 Member 1), EPB42 (Erythrocyte Membrane Protein Band 4.2), SPTB (Spectrin Beta. Erythrocytic) and ANK1 (Ankyrin 1) , four additional genes were found , all involved with immune response. Furthermore, two genes, the CCL26 and SLC4A1, have not been enriched in the gene network. CCL26 (chemokine (C-C motif) ligand 26) is from the cytokine family of secreted proteins involved in immunoregulatory and inflammatory processes, displaying chemotactic activity for normal peripheral blood eosinophils and basophils, and it may contribute to the eosinophil accumulation in atopic diseases. Finally, the SLC4A1 gene encodes a protein that is part of the anion exchanger family and is expressed in the plasma membrane of erythrocytes, involved in the transport of carbon dioxide from the tissues to the lungs. Furthermore, this gene is located in QTL regions previously described as associated with femur mineral content and femur weight, which reinforces its importance as a good candidate gene to FHS.In addition to the identification of the DE genes, biological processes and SNPs found in our study, another interesting result presented here was the comparison of the chicken transcriptomes with other datasets. When we compared the DE genes obtained in the AC and GP tissues with other gene expression datasets obtained in the GEO, we found four genes in common, kyrin 1) A, all ofBased on the functions and the high expression of the genes that were DE in both tissues, we had two hypotheses: the first is that the high expression of these genes may be a consequence of the FHS, due to cell damage and from the attempt to combat the progression of this condition. The second hypothesis is that the high expression and possible mutations of these genes cause FHN, as they can alter the structure of the actin cytoskeleton, affecting the structural integrity of the femoral articular cartilage, contributing to the occurrence of FHS. Therefore, using the two datasets in this study, we could highlight common genes that were regulated in the two tissues, as well as those that were exclusively DE in each one. With this information, new genes involved with FHS in broilers were described, where most of them have never been cited as candidates to this condition in chickens. We have also shown conserved mechanisms among huma and chicken osteonecrosis. Furthermore, genetic variants were identified; some of them were described for the first time in this study, and some of them differed between the normal and FHS-affected groups and could be further investigated as molecular markers for this condition. Finally, our results can also contribute to understand the onset of this condition in other species since the molecular mechanisms seem to be at least in part conserved among them.SLC4A1, RHAG, ANK1, MKNK2, SPTB, ADA, C7 and EPB420 genes should be more explored in order to validate them as candidates for FHS/FHN.With the integrated analysis of gene expression of the two tissues and variant identification, we were able to detect strong candidate genes and SNPs related to FHS/FHN in chickens. Comparing the AC and GP DE genes with the other datasets related to FHN allowed us to identify genes possibly involved with FHN, evincing the shared mechanisms across different species. Finally, the role of


Movimente Intervention study were analyzed (n = 812 / mean age 13.0 years (sd 1.04). Data on SB hours per day, PA minutes per week and weekly consumption frequencies of fruits, vegetables, salty snacks, candies and soda were self-reported on the validated Movimente questionnaire. Classes of healthy and unhealthy behaviors were derived by latent class analysis. Logistic regression analysis was used to estimate the associations between adolescents’ weight status and classes. Results: two classes were identified for the whole sample and for boys and girls. All classes had high probabilities of engaging high time in SB. Male adolescents in the unhealthy class had low probabilities of being active and high probability of consuming a low-quality diet. In contrast, girls’ healthiest profile presented lower probabilities of being active compared to boys’ healthiest profiles. No association was found between weight status and classes. Conclusion: All classes had at least one unhealthy behavior, for both the whole sample, and for girls and boys. Girls’ profiles were unhealthier compared to boys’ profiles. Hence, it is recommended that intervention strategies to change behaviors need to be distinct according to sex, targeting more than one obesogenic behavior at the same time.Background: the relationship between behavior clusters and weight status, mainly in low- and middle-income countries, remains unclear. This study aimed to examine the association between profiles of physical activity (PA), diet and sedentary behavior (SB) with weight status in adolescents from a southern Brazilian city, according to sex. Methods: data from the  The prevalence of obesity has increased 20% during the past three decades, based on data from 195 countries ,4,5. Thun = 2084, 45% male) as being allocated in a cluster labelled inactive, high quality of diet [Review studies on the clusters of physical activity (PA), diet and SB in youths reported that most children and adolescents presented mixed patterns, exhibiting one or two healthy behaviors combined with one or no unhealthy behaviors ,7. Patte of diet . Similar of diet . A Belgi of diet . In addi of diet ,13,14,15 of diet . In addi of diet , and alsn= 5364), three dietary patterns were derived by latent profile analysis , and none of these patterns were associated with overweightness or obesity for either boys or girls [There are inconsistent findings regarding the association between behavior patterns and weight status. Some studies identified clusters characterized by either high SB and low levels of PA that are positively associated with being overweight ,18,19, wor girls .Movimente study in a Southern Brazilian city. The findings of this study will serve as a basis to assess the effectiveness of the intervention conducted by the Movimente study, and can contribute to the development of multicomponent interventions to increase PA levels, decrease SB and improve the quality of diet at the same time among adolescents. Furthermore, these data will aid to identify adolescent groups at higher risk of overweight and obesity, as well as to better understand how SB, PA and diet cluster together, as potential targets for interventions related to the school environment.The majority of studies addressing cluster or latent class analysis on adolescent behaviors related to PA, diet and SB were conducted in high-income countries, and few studies evaluated these factors according to sex ,15. In oMovimente Intervention Program, carried out in 2017. This program aimed to increase PA and decrease SB among adolescents. The study was approved by the Federal University of Santa Catarina Ethics Committee (Protocol number 1.807.825 in 7 November 2016). The intervention was registered in the Clinical Trials Registry (NCT02944318), with the approval of the Brazilian National Research Ethics System (protocol number: 1.259.910).This is a cross-sectional study using baseline data from the Movimente Program is a school-based cluster randomized controlled trial on adolescents (grades 7th to 9th). Information on intervention protocol, sample size and randomization can be found elsewhere [n = 1427). In total, 999 adolescents provided written informed consent, and had free informed consent from parents (answer rate: 70%). Once students provided consent to participate in the study, they answered the Movimente questionnaire during regular school hours and anthropometric data were collected by trained professionals.The lsewhere . All stuIn this study, 921 students answered the questionnaire at baseline, and 859 and 861 had data on weight and height, respectively.PA was assessed using the Self-Administered Physical Activity Checklist previously validated for Brazilian adolescents . AdolescSB was assessed with four questions previously validated for Brazilian adolescents . Adolescp < 0.05 and p > 0.60 were acceptable, respectively [Using a standardized questionnaire ,25, adolectively . To obtaectively . Factor ®, Belo Horizonte, Brazil), with the subject standing up straight with feet together, head in Frankfort plane and arms hanging freely. Weight was measured using an electronic scale to the nearest 0.1 kg .Height and weight were measured based on international standardization . AnthropBody mass index (BMI) was computed as weight (kg)/height (m²). Age and sex-specific BMI z-scores were calculated according to the World Health Organization (WHO) . Weight Sociodemographic variables included sex, age (years) and maternal level of education . The students reported sociodemographic variables.Participants’ characteristics were described using means and standard deviations for quantitative variables and relative and absolute frequencies for qualitative variables. Comparisons of prevalence of overweight, including obesity vs. non-overweight between classes of behavior and between sexes, were performed using the chi-square test.Latent Class Analysis (LCA) was performed in Software R, using the poLCA package. LCA is an optimal strategy for examining varying groupings based on an individual’s patterns of responses to multiple categories variables . Latent Statistical analyses for association were conducted using Stata Statistical software, version 15 . Logistic regression adjusted by age and maternal education was used to verify the association of clusters with weight status . Values were expressed as odds ratio (OR) and 95% confidence intervals (95% CI). The least healthy cluster was the reference category.n = 812 ), and association analysis was performed for 79.4% (n = 731). From 921 students that answered the questionnaire, 109 students did not report some behavior variables , 140 did not have weight and/or height measurements and 115 did not report maternal level education. Latent class analysis was performed for 88.7% of the sample  was the unhealthiest, and was mainly characterized by the high probability of practicing PA <300 min per week, spending >4 h per day engaged in SB, consuming F&V <2 times per week and SSS >2 times per week. On the other hand, class 2 (43.84% of the students) were characterized by the high probability of practicing PA ≥420 min per week, spending >4 h per day engaged in SB and consuming >4 and 1–2 times for week of F&V and SSS, respectively.Boys. Class 2 was composed by 50.52% of boys, who had high probabilities of practicing PA ≥420 min weekly, consuming F&V >4 times per week, spending >4 h per day engaged in SB and consuming 1 to 2 times per week of SSS. Class 1  comprised 49.48% of boys whose behaviors were characterized by low probabilities of practicing PA ≥420 min weekly and consuming F&V >4 times per week. In addition, they had high probabilities of spending >4 h per day engaged in SB, and consuming SSS >2 times per week.Girls. Class 2 included a higher proportion of girls (65.26%), compared to Class 1. Girls in this class had high probabilities of practicing ≥420 min per week of PA, spending >4 h per day of engaged in SB and consuming SSS >2 times per week. Members of this class also presented low probabilities of consuming F&V >4 times per week. Class 1 comprised 34.74% of girls, which was characterized by higher probabilities of practicing <300 min per week of PA, spending 2 to 4 h per day engaged in SB, consuming >4 per week of F&V and >2 and 1 to 2 times per week of SSS.This study examined the patterns of PA, diet and SB and assessed its associations with weight status among Brazilian adolescents. Due to country-specific differences in food intake, PA and SB, the present study provides valuable information on clusters of these behaviors among adolescents from an upper-middle-income country. Furthermore, data from high-income countries showed that the prevalence of overweight and obesity among young people have plateaued. In contrast, data from medium- to low-income countries indicates that the prevalence is still rising .Two classes were identified for all stratum (whole sample and for boys and girls). All classes had high probabilities of engaging high time in SB. No association was found between weight status and clusters. The unhealthiest profile (Class 1) was more prevalent among the participants, which is in line with previous studies ,11. ClasMore boys were allocated in the healthiest profile (Class 2) characterized by high time practicing PA and weekly frequency of consuming fruits and vegetables. The boys in the unhealthiest class had low probabilities of being active and consuming a low-quality diet. In contrast, the girls’ healthiest profile presented low probabilities of being active compared to boys’ healthiest class, corroborating with data from the literature ,7,12,13.Studies have identified that girls had the unhealthiest profiles and a higher presence in clusters with low levels of PA compared to boys ,13. ThesThese findings enforce the importance of interventions to promote PA and the consumption of healthy foods, as well as reduce SB in youths , once thIt is difficult to make direct comparisons between the present results and those of other studies, due to the different analytical methods used to derive clusters behaviors among adolescents, such as cluster k-means ,14,19 anOur results unexpectedly found no association between clusters and weight status, a finding that is not unique ,13,14,15There are limitations that must be considered when interpreting the study results. Data was self-reported, which has well-known disadvantages , and make it impossible to explore the domain of variables, intensities of PA and energy intake, although all questionnaires used were validated. Objective measurements, such as an accelerometer, are more accurate to determine the amount of PA and SB compared to questionnaires . HoweverThe classes of PA, diet and SB in Brazilian adolescents can be described in two profiles characterized by at least one unhealthy behavior, with all classes presenting high probabilities to spend more than 2 h engaged in SB. No association between classes and weight status was observed. Once the classes were predominantly characterized as unhealthy behaviors and the obesity etiology is multifactorial, results suggest the development of multicomponent strategies, which focus on a wide range of obesogenic behaviors, enable the shift to a healthier lifestyle. Thus, programs should consider different actions according to sex, promoting PA for girls and a balanced diet and SB for both sexes.


Myasthenia gravis (MG), an acquired autoimmune-related neuromuscular disorder that causes muscle weakness, presents with varying severity, including myasthenic crisis (MC). Although MC can cause significant morbidity and mortality, specialized neuro-intensive care can produce a good long-term prognosis. Considering the outcomes of MG during hospitalization, it is critical to conduct risk assessments to predict the need for intensive care. Evidence and valid tools for the screening of critical patients with MG are lacking. We used three machine learning-based decision tree algorithms, including a classification and regression tree, C4.5, and C5.0, for predicting intensive care unit (ICU) admission of patients with MG. We included 228 MG patients admitted between 2015 and 2018. Among them, 88.2% were anti-acetylcholine receptors antibody positive and 4.7% were anti-muscle-specific kinase antibody positive. Twenty clinical variables were used as predictive variables. The C5.0 decision tree outperformed the other two decision tree and logistic regression models. The decision rules constructed by the best C5.0 model showed that the Myasthenia Gravis Foundation of America clinical classification at admission, thymoma history, azathioprine treatment history, disease duration, sex, and onset age were significant risk factors for the development of decision rules for ICU admission prediction. The developed machine learning-based decision tree can be a supportive tool for alerting clinicians regarding patients with MG who require intensive care, thereby improving the quality of care. Myasthenia gravis (MG) is an acquired autoimmune neuromuscular disorder presenting as muscle weakness in the eye, bulbar, limbs, and respiratory muscles that worsens with repeated muscle motion ,2. The pAlthough MC can cause significant morbidity and mortality, specialized neuro-intensive care, including respiratory monitor, nutrition support, non-invasive positive pressure ventilation, and intubation, can produce a good long-term prognosis in patients with severe MG symptoms ,13,14. TAutoimmune diseases, including MG, have chronic and fluctuating courses along with a complex pathophysiology. Prediction of outcomes and risk factors for autoimmune diseases is difficult due to the different phenotypes of clinical presentation. Current medical treatment integrates machine learning (ML) methods that play a critical role in personalized medicine by providing computers with the ability to learn from experience without rules specified by humans . The basConsidering the heterogeneity of myasthenia crises and the importance of intensive care to improve prognosis, it is critical to conduct risk assessments to predict the need for intensive care. Few studies had identified the risk factors of requirement of intensive care or intubation in patient with MG, including higher MG activities of daily living (MG-ADL) scores, initial symptom of bulbar weakness, infection, higher PCO2, and higher Myasthenia Gravis Foundation of America (MGFA) clinical classification ,23. HoweThe data of 513 hospital admissions of patients with MG who were admitted to the Shin-Kong Wu Ho-Su Memorial Hospital in Taipei, Taiwan, between December 2015 and October 2018, were retrospectively analyzed. The inclusion criteria for patients with MG were (1) admission due to MG symptoms deterioration and (2) admission for MG-related management, including thymectomy or for immunotherapy. The exclusion criteria were (1) incomplete data and (2) admission that unrelated to myasthenia gravis. A total of 188 hospital admissions were excluded because they were not due to MG and 13 were excluded due to data loss, respectively. After cleaning the data, data from 200 patients with 312 hospital admissions were used for the analyses. The data in cases where the same patient had been hospitalized for the same reason were merged. Finally, a total number of 228 hospitalizations (including 200 patients) were used for the analysis . The proA retrospective review of medical records, including information on the age, sex, age at diagnosis, disease duration, autoantibodies present, medications used, maximum dosage of corticosteroid before admission, thymic histology, history of thymectomy, treatment during hospitalization, and length of ICU admission, was conducted. Disease severity was graded according to the classification of the Myasthenia Gravis Foundation of America (MGFA) classification. Twenty clinical variables were collected. The inclusion criteria for patients with MG were (1) Myasthenia Gravis Foundation of America (MGFA) class II and III, and (2) no medication adjustment in the last 6 months. The exclusion criteria were (1) unstable MG symptoms and (2) history of intensive immuno-modulation therapy, including immunoglobulins, high dose intravenous corticosteroid, or plasmapheresis, 6 months before enrollment—because the use of these short action immunotherapy means that the patient has a life-threatening phenomenon and unstable symptoms. Patients were eligible if they were diagnosed with MG based on the MGFA criteria. Briefly, the diagnosis of MG was based on fluctuating muscle weakness with fatigability, decreased symptom severity after use of acetylcholinesterase inhibitors, decremental changes in repetitive nerve stimuli on repetitive nerve stimulation test, or presence of anti-AchR autoantibodies.The MGFA clinical classification was based on previous reviews that represented the clinical severity of the patient upon admission . The maxThe history of thymectomy was divided into three categories, as follows: (1) the patient had never undergone thymectomy; (2) the patient had undergone thymectomy during this admission; (3) thymectomy had been performed previously. The treatment during hospitalization included plasmapheresis, intravenous corticosteroid administration, intravenous immunoglobulin administration, and rituximab administration. Treatment with plasmapheresis was divided into three categories, as follows: (1) the patient did not undergo plasmapheresis; (2) the patient underwent five sessions; (3) the patient underwent more than five sessions. The serology status of MG autoantibodies included anti-AChR antibody and anti-MuSK antibody positivity or negativity, as well as double seronegativity.As per the protocol of our hospital, patients with MG who are hospitalized for thymectomy must be observed in the ICU for 1 day after the operation. This distinguishes which groups of MG patients require ICU admission and then further divides them into two categories: ICU admission more than 1 day and less than 1 day. Most patients with MG are treated regularly in the ward, and those who require thymectomy are required for admission to the ICU for 1 day. Therefore, ICU admission in our study was defined as greater than 1 day.In total, 228 patients included in the study  along wiThe MGFA clinical classification at admission divided the patients into 5 groups: 24 patients (10.53%) were classified as class I, 88 patients (38.60%) as class II, 74 patients (32.46%) as class III, 26 patients (11.40%) as class IV, and 16 patients (7.02%) as having MG crisis. Regarding the medications used, 141 patients (60.1%) were treated with different oral immunosuppressants. According to the thymus histology, 110 patients (48.25%) had thymoma, and 67 patients (29.39%) had thymic hyperplasia. A total of 148 patients underwent thymectomy.ML-based decision tree algorithms are popular and effective approaches for clinical/healthcare classification problems that visually represent the decision rules of generated predictions using a tree-shaped figure ,26,27. ADuring the training phase, the hyperparameters of CART, C4.5, and C5.0 methods needed to be tuned for constructing a relatively well performed model, so we applied a 10-fold cross-validation technique for tuning the hyperparameters of each method. The training dataset was further randomly divided into 10 equal-sized folds, then 9 folds were used as a training dataset to build the model with a different set of hyperparameters, the remaining 1 fold was used as a validation dataset for model validation. The 10-fold cross-validation was repeated 10 times, by changing the fold of the validation dataset, thus the best hyperparameters set, which generated the best validation performance, based on the area under the receiver operating characteristic (ROC) curve (AUC) metric for each decision tree method, which was then used to construct the best CART, C4.5, and C5.0 models.In the model evaluation phase, the testing dataset was used to evaluate the predictive performance of the best CART, C4.5, and C5.0 models. The metrics used for performance comparison were accuracy, sensitivity, specificity, AUC, and F1 score. In order to provide a more robust comparison, the process mentioned above was randomly repeated 10 times, and the averaged metrics were used to find the best decision model among the best CART, C4.5, and C5.0 models.Finally, based on the best decision tree model, the decision rules could be developed, and the important clinical variables were identified. The rules were then discussed to improve the early recognition and care of patients with MG who need intensive care.All methods were implemented in the R software with the 3.6.2 version. LR was constructed by the “blorr” package with the 0.3.0 version ; CART waAccording to the proposed scheme, for modeling effective CART, C4.5, and C5.0 models, the hyperparameters of each method were tuned and evaluated. The LR method without hyperparameters tuning—the baseline method—was constructed by using the proposed scheme. The values of the hyperparameters which generated the best CART, C4.5, and C5.0 models with the highest AUC values are listed in . The figure shows that C5.0 is the best predictive model in this study.The performance of the LR, CART, C4.5, and C5.0 methods with 10 repetitions is shown in Figure 4As C5.0 has the best AUC and outperforms the four competing methods, the important clinical factors and decision rules generated and suggested by the best C5.0 model are discussed.This is the first study to use the ML decision tree method for predicting ICU admission in patients with MG. The C5.0 method generated the best and most promising classification results and provided an output of six clinical features that were critical for determining the risk of ICU admission. The MGFA classification and the presence of thymoma were two of the important physiological indices. The MGFA clinical classifications are used to identify the different clinical features and severity of patients with MG . The higIn addition, this study demonstrated that some treatments have influenced ICU admission in patients with MG, including treatment with oral azathioprine (AZA). According to the international guidelines for the management of MG, immunosuppressive therapy is used in patients with MG who have poor response to pyridostigmine alone . AZA wasThe onset age and disease duration also influenced ICU admission in patients with MG. Our results showed that late-onset MG, defined as MG at an onset age >50 years, had a negative prediction for ICU admission. Previous studies indicated that patients with late-onset MG are likely to have a thymoma and a severe disease that is difficult to treat ,46. An oOur findings found that disease duration shorter than 41 months was a factor that could influence ICU admission in patients with MG . Many reManagement of MG upon admission would benefit from a good understanding of the disease course. In this study, many of the risk factors associated with the prognosis of hospitalized MG patients requiring ICU admission were identified using the C5.0 algorithm. C5.0 is a promising ML-based decision tree algorithm and has been successfully used in clinical/healthcare issues ,53. PrevSome investigators have evaluated the potential risk factors of ICU care in patient with MG. A retrospective study showed that higher MG-ADL scores with bulbar involvement and higher MGFA classification were associated more frequently in the ICU group. Better outcomes may be obtained with early intensive care management if the patient presents with those factors . Liu et This study had several clinical implications. First, the variable phenotype of MG makes it difficult to determine the optimal management plan and prognosis; therefore, physicians can use this decision tree model to identify patients likely to have ICU admission during hospitalization. Second, this study may provide a possible tool for clinical guidance for ICU clinicians to improve intensive care quality. This is because patients with MG who are likely to stay in the ICU have a high mortality, require a ventilator, and suffer severe disability. Due to the improvement in management and development of intensive care techniques, the fatality rate of MG has been less than 5% in the recent years . An earlThere are some limitations to this study. First, the usefulness of the model is probably restricted to our hospital because of inter-hospital differences that impact the model of ICU strategy in MG. Second, the data were acquired from chart reviews, and the specific details of each patient, such as the quantitative MG score, which can quantify the severity and outcomes of treatment, were not available for analysis. Third, due to the heterogeneity of MG symptoms, our model failed to account for additional clinical factors known to influence ICU admission. These include the partial pressure of carbon dioxide at admission, the activities of daily living score at myasthenia crisis onset, and nosocomial infection ,20. FinaThis study uses an ML-based decision tree approach to predict ICU admission in patients with MG. Decision tree methods are promising tools that can build a predictive model of ICU admission in patients with MG, and they can provide physicians with information to evaluate the potential risk of ICU admission in patients with MG. Due to the varying clinical presentation of patients with MG, the model produced can be used for performance benchmarking and as a supportive tool for alerting clinicians regarding the patients with MG who require intensive care and management; therefore, enabling clinicians to provide timely and effective treatment, improving care quality and patient outcomes.


The aim of this study was the anatomical description of the orbital region, eye tunics, upper and lower eyelids, superficial gland of the third eyelid with the third eyelid, and lacrimal gland of the Asiatic black bear. The eyeball morphometry, the orbital region analysis, macroscopic, morphometric and histological analysis of the eye tunics and anatomical analysis of the accessory organs of the eye with histochemical methods were used. The anatomical structures within the orbital region of Asiatic black bear were typical of only the family Ursidae and were similar to the family Canidae. The macroscopic and histological structure of the eye and eyelids was comparable to the structure of the eye and eyelids in other representatives of the Ursidae family, although features typical of only Canidae or terrestrial Mustelidae and Pinnipedia were also observed. The Bowman’s membrane of the cornea was absent, similar to all domestic dogs and some wild dogs. The pupil was similar to other bear species subfamilies, Tremarctinae and Ursinae, and also to domestic and wild dogs. The lens was biconvex round, similar to the Canidae. The retina was composed similarly to the diurnal terrestrial carnivores. In both eyelids were observed very well-developed tarsal glands, ciliary glands and sebaceous glands. The third eyelid was T-shaped and composed of the hyaline tissue and contained conjunctival-associated lymphoid tissue (CALT), which was also the case in Canidae. The obtained results indicate that the features of the anatomy of the eye and orbital region in the Asiatic black bear are also typical of the Ursidae family. In this study, we present first data concerning the morphological observations of the orbital region, eye tunics, upper and lower eyelids, superficial gland of the third eyelid with the third eyelid, and lacrimal gland in captive adult male Asiatic black bear. The following research methods were used in the work: the eyeball morphometry, the orbital region description, macroscopic description, morphometric and histological analysis of the eye tunics and selected the accessory organs of the eye , Methyl-green-pyronin Y (MGP Y), Movat pentachrome, and picro-Mallory trichrome) as well as histochemical examination . The eyeball of the Asiatic black bear was a spherical shape, while the periorbita was funnel/conical-shaped and the eye socket was of the open type. The cornea was absent of the Bowman’s membrane similar to all domestic dogs and some wild dogs. There were palisades of Vogt in the corneal limbus epithelium similar to the Canidae. Degenerative choroidal tapetum lucidum similar to ranch mink (Mustelidae) has been found. The pupil was big and round in shape. The ciliary muscle, dilatator and sphincter muscle were well developed, similar to the pinnipeds. The lens was biconvex round, similar to the Canidae. The retina was composed similarly to the diurnal terrestrial carnivores. In both eyelids were observed very well-developed tarsal glands, ciliary glands and sebaceous glands. The orbital zone in the eyelids was characterized by lymphoid follicles, diffuse lymphocytes and specialized high endothelial venules. In the anterior palpebral margin of the upper eyelid, soft and short eyelashes were observed, while in the lower eyelids they were absent. The third eyelid was T-shaped and composed of the hyaline tissue, and it contained CALT, similar to that in Canidae. The superficial gland of the third eyelid was a multilobar alveolar branched complex with seromucous nature, while the lacrimal gland was also a multilobar acinar branched complex gland, but producing a mucous–serous secretion. The results of our research indicate that the features of the anatomy of the eye and orbital region in Asiatic black bear are also typical of the Ursidae family. Moreover, a detailed analysis of the morphological eye region may be useful in comparative studies and veterinary diagnostics in this bear species. Ursus thibetanus), also known by another name as the moon bear or the ring bear, was previously known as Selenarctos thibetanus. It occurs in seven subspecies [The Himalayan bear , brown bear (Ursus arctos), leopard (Panthera pardus), and sometimes wolves (Canis lupus) and Eurasian lynx (Lynx lynx) [The  on them . They bu on them . In the nx lynx) . These animals use all possible food sources, including agricultural produce and livestock, and are therefore often killed at the hands of farmers ,8,9. AnoAccording to Brown, 2009 , and HunThere are currently 68 institutions (zoos) housing Asian black bears, in which there are 257 individuals on 3 continents . In Europe, there are 61 individuals in 29 zoos , while in Poland, only 4 individuals are found in 3 zoos—Wroclaw , Toruń  and Chorzów  (date from 16 October 2021) (Species 360.org).Ursus arctos has quite adequate visual capacities. Kuckuk, 1937 [Ursus maritimus eyes appear black but are a golden brown; sometimes they are greenish-yellow in the dark), being blue at birth ; large polar bears are an exception; the pupils of bears are round, except for those of the giant panda Ailuropoda melanoleuca, which are vertical slits, possibly for improved night vision, and are widely spaced and face forward [The first descriptions of eyesight in the bear appeared at the beginning of the 20th century ,13,14,15uk, 1937 , remarksuk, 1937 , the eyeuk, 1937 . However forward , describ forward . In addi forward . In addi forward , reportsUrsus americanus [Ursus thibetanus [Ursus arctos horribilis [Ursus arctos [Melursus ursinus [Ursus malayanus [Ursus maritimus [The literature describing the correct structure of the skull while taking into account the anatomical description of the orbital region, the eyeball and the accessory organs of the eye at the macro and microscopic level in the family Ursidae  and The Journal of Laws of the Republic of Poland, the Act of 15 January 2015, on the protection of animals used for scientific or educational purposes. Personal permits issued by the District Veterinary Doctor in Wrocław (Poland) .n = 2), upper eyelids (n = 2), lower eyelids (n = 2), superficial gland of the third eyelid (n = 2), third eyelid (n = 2) and lacrimal gland (n = 2). Muscle of eyeball, orbital fat body, and fascial sheath of eyeball have been partially removed for microscopic examination from eyeballs. Nomina Anatomica Veterinaria (2017) [Nomina Histologica Veterinaria (2017) [Axial eye diameter: from the corneal vertex to the root of the optic nerve;Maximum transverse  eye diameter;Minimum transverse  eye diameter;Maximum and minimum corneal diameter;Corneal axial thickness and corneal peripheral thickness;Lens axial length;Aqueous chamber depth;Vitreous chamber depth;Tapetum lucidum length;Tapetum lucidum thickness.The following were collected for the research: eyeballs (a (2017)  and Nomia (2017)  were usea (2017)   [Morphometric measurements of eyeballs (see points a–j), eyelids and orbital glands  were measured using a digital caliper . Due to the analysis of only these Asiatic black bears, no statistical tests were used in the following work. The values of six randomly measurements of the whole eyeballs, eyelids and orbital glands were recorded. The obtained measurements were analyzed statistically: mean and standard deviation (S.D.). The anatomy of the orbital region of the Asiatic black bear was analyzed based on the anatomical description of the orbit by Nickel et al., 2004 , and Noma (2017) .Immediately after the death of the animal, the eyeballs with selected additional organs of the eye were subjected to preparation, morphometry, and then the collected samples were placed in 4% buffered formaldehyde for at least 72 h and then rinsed in running water for 24 h. Then, the samples were processed in a vacuum tissue processor—ETP  and embedded in paraffin. The specimens were cut using a Slide 2003  sliding microtome into 4 µm sections. The following histological stains were performed:Fontana–Masson: for visualization of melanin (black color);Mayer’s hematoxylin and eosin: for general histological description;Picro-Mallory trichrome: for detection of different components of connective tissue ;Methyl-green-pyronin Y: for demonstration of plasma cells ;Movat pentachrome (modified Russell Movat): for demonstration of collagen and reticular fibers (yellow color), elastic fibers (black to blue/black color), muscle (red color), mucin (bright blue color) and fibrin (bright red color).The slides obtained were then observed using the Zeiss Axio Scope A1 light microscope  and rated a scoring system based on a standard protocol previously described ,59,60. TPeriodic acid-Schiff (PAS): for identification of glycans, glycoconjugates, neutral or weakly acid glycoproteins (magenta color); Alcian blue pH 1.0 (AB pH 1.0): for visualization of strongly sulfated mucosubstances (blue color);Alcian blue pH 2.5 (AB pH 2.5): for demonstration of acid mucopolysaccharides (dark blue color);Alcian blue pH 2.5 PAS (AB pH 2.5/PAS): for identification of acidic sulfated mucosubstances and sialomucins (blue color) and neutral mucins (magenta color);Hale’s dialyzed iron (HDI) for visualization of carboxylated and sulfated mucopolysaccharides and glycoproteins (deep blue color). All histochemical stains were performed based on the following analyses: Bancroft and Gamble, 2008; Carson, 1990; Munakata et al., 1985; Sheehan and Hrapchak 1980 ,63,64,65The eyeball. The eyeball in the Asiatic black bear has a spherical shape. The axial eye diameter was 16.7 ± 0.4 mm, approximately. The maximum transverse eye diameter was 17.127 ± 0.09 mm, and the minimum transverse eye diameter was 18.143 ± 0.08 mm, approximately. The maximum corneal diameter was 12.404 ± 0.2 mm, and the minimum corneal diameter was 11.56 ± 0.2 mm, approximately. The scleral (limbus) thickness was 1.315 ± 0.09 mm approximately, while the scleral (equator) thickness was 0.903 ± 0.3 mm, approximately and the scleral (optic nerve) thickness was 0.61 ± 0.09 mm, approximately. The corneal axial thickness was 0.851 ± 0.04 mm, and the corneal peripheral thickness was 0.835 ± 0.05 mm, approximately. The aqueous chamber depth was 2.013 ± 0.6 mm, while the vitreous chamber depth was 5.578 ± 0.1 mm and the lens axial length was 6.305 ± 0.08 mm, approximately. Macroscopic examinations revealed the presence of a very faintly marked tapetum lucidum which resembled a crescent moon with a dark brown color, being slightly opalescent. The length of tapetum lucidum was 8.063 ± 0.05 mm, while the width of tapetum lucidum in the narrower part was 3.573 ± 0.08 mm, and in the wider part was 6.719 ± 0.1 mm, approximately.The orbital region. The periorbita in the Asiatic black bear was funnel/conical-shaped and the eye socket was of the open type. The orbital ring was filled with an orbital ligament connecting the very high frontal process of the zygomatic bone with clearly marked the zygomatic process of the frontal bone. The bones included in the orbital region were: an orbital part of the frontal bone, a small and narrow facial and orbital surface of the lacrimal bone, a large sphenoidal process of the palatine bone and a large pterygoid process of basisphenoid bone. The orbital surface of a body of maxilla was marked and it was in contact with the pterygopalatine surface, where a large maxillary foramen was located intranasally, which passed through a very short infraorbital canal (about 1.3–1.4 cm) into the infraorbital foramen located at the height of the 3rd tooth cheek. There were two smaller holes on the perpendicular plate of palatine bone: sphenopalatine foramen, and palatine caudal foramen directly behind it. On the facial surface of the lacrimal bone in the nasal direction, there was a small prominence—rostral lacrimal process—and on the orbital edge of the lacrimal bone, there was a large millet grain size—caudal lacrimal process. The supraorbital foramen, trochlear fovea for the superior oblique muscle and fossa of ventral oblique muscle were absent in the Asiatic black bear. The fossa for the lacrimal sac, which lead through a single lacrimal foramen to the lacrimal canal, was present on the orbital surface of the lacrimal bone. The temporal surface of the frontal bone had a sharp and very clearly exposed orbitotemporal crest. Directly below the above-mentioned crest was a single medium-sized ethmoid foramen for the n. Ethmoidalis. About 1.3–1.4 cm from the ethmoid foramen was the optic canal for the optic nerve, which ran within the wings of the presphenoidal bone. Then, about 2.1–2.2 cm from the optic canal, above the pterygoid crest, was located a large orbital fissure, and directly below was also a large foramen rotundum, which was present on the wings of the basisphenoid bone. The orbital fissure and foramen rotundum form the route for n. Ophthalmicus, n. Oculomotorius, n. Trochlearis, n. Abducens and n. Maxillaris. Downstream of the foramen rotundum was the rostral alar foramen, which led via the alar (pterygoid) canal (approximately 1.2–1.3 cm long) to the caudal alar foramen F–I.The upper and lower eyelids. In the anterior palpebral margin of the upper eyelid, soft and short eyelashes were observed, while in lower eyelids they were absent A. The paThe superficial gland of the third eyelid. The superficial gland of the third eyelid was small (approximately 8.241 ± 0.2 mm long × 10.44 ± 0.4 mm wide × 4.815 ± 0.2 mm thick) (was wider than longer), oval in shape and had a light pink color B. These The third eyelid. The marginal part of the third eyelids was very pigmented (brown-black color) and was thick. The third eyelid was T-shaped and consisted of an upper and lower branch (the length of the upper and lower branch together was approximately 14.003 ± 0.5 mm) and a crossbar (length was approximately 8.347 ± 0.3 mm) B. The thThe lacrimal gland. The lacrimal gland was very small (approximately 8.819 ± 0.2 mm long × 5.2 ± 0.1 mm wide at its widest point × 3.596 ± 0.2 mm thick), triangular in shape and was light pink in color C. It wasstratum pigmentosum, stratum bacillorum et conorum, membrane limitans glie externa, stratum granulosum externum, stratum plexiforme externum, stratum granulosum internum, stratum pexiforme internum, startum fibrarum nervosarum et membrana limitans glie interna ; the middle layer, defined as the proper substance of sclera consisted of collagen fibers and weakly marked elastic fibers that formed distinct lamellae, arranged to the eyeball surface, sparse granules of melanin clusters, sparse blood vessels; the thin dark lamina of sclera layer, being in close contact with the suprachoroid layer and composed of fibroblasts, few blood vessels and melanin granules A. The co interna H. The re interna H. The thThe upper and lower eyelids. The anterior surface of the upper eyelid and lower eyelid was covered by stratified squamous epithelium with five to eight layers of nucleated cells. The superficial layer of the stratified squamous epithelium was covered with a thick stratum corneum (57.369 ± 17.1 µm). The stratum basale epithelial cells lying on the basement membrane contained a small number of melanocytes. Under the epithelium lay a thick lamina propria formed by loose fibrous connective tissue, which was characterized by the presence of numerous elastic and reticular fibers A. Well-dThe superficial gland of the third eyelid. These glands are characterized by a follicular multilobar branched complex structure with seromucous nature. This gland was surrounded by a big intraperiorbital fat body, under which was a thick connective tissue capsule. From this connective tissue capsule, both thick and thin interlobar septa, which divided gland structure into numerous big and small lobes, departed deeper into the gland A. The coThe third eyelid. The palpebral conjunctiva of the third eyelid was covered by a multilayer squamous epithelium composed of five–eight layers of nucleated cells and numerous goblet cells, while the bulbar conjunctiva of the third eyelid contained a multilayered cubic epithelium consisting of three to four layers of epithelial cells with single goblet cells A–C. The The lacrimal gland. These glands were a multilobar acinar branched complex structure producing a mucosal–serous secretion. This gland was surrounded by a thick connective tissue capsule, interspersed with numerous fat cells, collagen and reticular fibers, which, penetrating deep into the gland, formed poorly marked flaps through narrow interlobar septa A. The exThe upper and lower eyelids. In both eyelids, there was a negative PAS, AB pH 1.0, AB pH 2.5, AB pH 2.5/PAS and HDI reactions in the tarsal and sebaceous glands see . The cilThe superficial gland of the third eyelid. The serous acinar cells showed a PAS and AB pH 2.5 weakly (−/+) reaction, AB pH 1.0 and HDI negative (−) reaction, while AB pH 2.5/PAS (blue color) weakly (+) positive reaction. For mucous acinar cells of these gland, PAS, HDI and AB pH 2.5/PAS (magenta color) middle positive (++) reaction, and AB pH 2.5 slightly (−/+) positive reaction were found . The AB The third eyelid. The goblet cells of the third eyelid showed a strongly (++/+++ or +++) PAS, AB pH 1.0, AB pH 2.5, AB pH2.5/PAS (magenta color) and HDI positive reaction . The lacrimal gland. Staining of the serous acinar cells using the PAS method demonstrated the presence of a weakly positive reaction (+). The AB pH 1.0, AB pH 2.5 and HDI staining techniques proved the presence of negative (−) reaction in these cells. The AB pH 2.5/PAS method revealed a blue reaction in the serous acini rated as ++. The numerous mucous acinar cells in these glands gave a positive reaction, which was rated as ++ using the PAS, AB pH 2.5, AB pH 2.5/PAS (magenta color) and HDI stains. These mucous cells characterized negative reaction using AB pH 1.0 stain . The Ursidae family arose early in carnivoran evolution from Miadicae—a small tree-climbing common ancestor (approximately 33–37 million years old)—and together with the Canidae, is thought to be one of the most ancient families within the Caniformia . CurrentUrsus americanus, grizzly bear Ursus arctos horribilis and sloth bear Melursus ursinus [Cerdocyon thous, Arctic fox Alopex lagopus and bush dog Speothos venaticus) compared to mongrel dogs, South African painted dog Lycaon pictus, wolves Canis lupus, gray fox Urocyon cinereoargenteus and red fox Vulpes, where the eyeball measurements were characterized by much higher parameters [Ailurus fulgens [Ursus americanus and Ursus arctos craniometry, bears’ diet, activity pattern, and speed increase in the range of 47–48 km/h, we see that these animals have small eyeballs. According to Davis, 2019 [Odobenus rosmarus, like Phocidae and Otariidae, is not capable of echolocation, but his eyeballs are ellipsoidal in shape and are extremally small both in absolute size and in relative size to the body compared to other pinnipeds, and are located dorsolaterally on the head, which limits the binocular vision [Phoca vitulina, harp seal Pagophilus groenlandicus and the Weddell seal Leptonychotes weddelli, the eyes feature a high degree of astigmatism in the air, which results from corneal curvature and much of it disappears when the eye is underwater [The obtained results of the eyeball macroscopic measurements performed in the examined Asiatic black bear were similar to the brown bear, American black bear  ursinus ,66, but rameters ,66,67,68 fulgens ,66,69 ar fulgens . Accordi fulgens ,72,73,74is, 2019 , in the is, 2019 . It is ir vision ,76. In tr vision , and Hanr vision , in the derwater . Jamiesoderwater , and Jamderwater  suggest derwater , and Walderwater , was incderwater , reporteUrsus americanus, Ursus arctos, Melursus ursinus) as well as in some wild Canidae species  or in Procyonidae  [Canis dingo and corsac fox Vulpes corsac [Canis dingo, Canis familiaris, Cerdocyon thous and Vulpes corsac [Canis lupus and Lycaon pictus were slightly higher [Canis dingo and Vulpes corsac), while higher values of these dimensions occurred in the domestic dogs and South African painted dogs [Enhydra lutra, where the eyeball is similar to those terrestrial mammals—it is almost spherical and the axial eye length is only a little shorter than the diameter, but the corneal curvature has very low convexity [Eumetopias jubatus is the cornea in the central part characterized by low convexity and with no truly flat corneal emmetropic window. Because the corneal curvature in the seals, sea lions and walrus is poorly marked and the central part of it is absent, the outer surface of the cornea is minimal and does not function as a refractive unit in air. Due to the small curvature of the cornea and the similar refractive index of the cornea and water, the cornea does not function as a refractive unit in water. Therefore, changes in the media in front of the cornea, from air to water or from water to air, have a minimal or no effect on the position of the focused image [The maximum and minimum corneal diameter in the examined Asiatic black bear was similar to the other Ursidae species (n lotor) . Histolos corsac ,85, whils corsac . In the s corsac , while Cy higher ,87,88. Nted dogs ,84. In tted dogs ,90, as wonvexity . As repoonvexity , of all ed image . It is iLeptonychotes weddellii consists of 5 layers and the corneal thickness described in pinnipeds was similarly  [Histologically the cornea in the Antarctic Weddall seal  walrus) ,91,92, a walrus) ,91.Our research showed, similarly to South African painted dogs, domestic dogs, cats, rats and humans within corneal limbus epithelium, the presence of palisades of Vogt, composed of conjunctival folds, containing niches for limbal epithelial stem cell (LESC) . The LESChrysocyon brachyurus, Canis lupus, Lycaon pictus pictus) [Ursus arctos horribilis the pupil had a wider temporonasal length, while vertically slit-shaped in the giant panda, Cerdocyon thous and Vulpes [Procyon lotor as nocturnal animals the pupil is wide [Ursus americanus, in which it is vertically oval, dilatating to a full circle. McKay Strobel et al., 2020 [Erignathus barbatus, which has a diagonal pupil, and the California sea lion, which has a pupil shaped like a tear-drop [In the examined terrestrial caniforms, the pupil was characterized by different shapes and for example, the pupil in our examined Asiatic black bear was big and round in shape, similar to that of other bear species subfamily Tremarctinae and Ursinae and also in the domestic dog and in some wild Canidae ( pictus) ,96, and  pictus) , the brod Vulpes ,86,96,97 is wide . Johnson is wide  reportedl., 2020 , reportel., 2020 , may posl., 2020 , reportsl., 2020 . Due to l., 2020  and Malml., 2020 . As repol., 2020 , most piear-drop . In the ear-drop . The Phoear-drop ,75. In tear-drop ,103,104,ear-drop . Howeverear-drop , in the Cercoleptes caudivolvulus (Procyonidae), the tapetum lucidum is a yellowish-green color, while on the ringtailed coati Nasua rufa (Procyonidae) it is bright green with yellowish patches [Mustela putorius it comprises 5 to 7 cell layers [The tapetum lucidum is a specialized reflective layer of the choroid, which is designed to increase the ability of the retina to function under low light levels and increase photoreceptor layer stimulation by intrinsic fluorescence of its structure when stimulated by incident light . Macrosc patches . The var patches , while i patches . Histolo patches ,111,112. patches , cited b patches , reporte patches ) the tap patches . In the  patches , while i patches ,111, in  patches , and in l layers ,111. OllThe lens of the eye is soft, transparent and was protein-rich . In the Ursus arctos horribilis, diurnal and nocturnal Canidae, and Mustelidae [Procyon lotor and kinkajou are monochromatic , while the coati Nasua nose is dichromatic .In our Asiatic black bear, the retinal photoreceptor layers consisted of rods and cones, as was seen in the brown bear stelidae ,83,114. stelidae , and Heystelidae , on the stelidae . The polstelidae . Accordistelidae ,119,120.stelidae  and Nagystelidae  on the pstelidae  on the hstelidae  showed tstelidae ,125. Resstelidae  on familMeles meles, marten Martes foin and otter Lutra lutra [Nyctereutes procyonides, and not present in our examined Asiatic black bear [Unfortunately, due to the presence of only one individual, we were not able to measure the orbit with craniometry and compare the obtained test results with the studies on other bear species, and therefore we focused only on the anatomical description of the orbital area. The detailed anatomical description of the orbital region in the examined Asiatic black bear, and Sloth bear , was simra lutra ,129, whiack bear . The opeack bear ,132,133.ack bear ,67,68. Aack bear , showed Scant or virtually no scientific reports on the morphology of the accessory eye organs in the Ursidae family, and also in parvorder Musteloidea, forced us to compare our results with the Canidae and parvorder Pinnipedia families.Mustela putorius furo [Macroscopic studies in the upper and lower eyelids in Asiatic black bear showed absent eyelashes in the anterior palpebral margin in the lower eyelid, similarly to the domestic dogs and wild dogs (South African painted dog and crab-eating fox) ,68,135, ius furo . Accordiius furo , the pinius furo . Collitzius furo , describius furo , points ius furo , in the ius furo , Moore eius furo , Moore aius furo , and Knoius furo , distribius furo . Our resius furo ,68,147. ius furo , the numius furo .Our studies have shown that this gland on the Asiatic black bear, just like in wild and domestic Canidae, is located in the medial corner of the eye, between medial straight and ventral straight muscles, and was partially covered by the ventral oblique muscles ,150,151.Procyon lotor lotor) and is a small orbital gland with mixed nature. Research conducted by Kastelein et al., 1993 [The third eyelid in the test Asiatic black bear was T-shaped, similarly and previously described in the domestic and wild Canidae, and it was very pigmented and located in the medial canthus of the eye ,158,159.l., 1993 , on the l., 1993  and Püttl., 1993 , where il., 1993 , this glCanis lupus familiaris, Cerdocyon thous or Lycaon pictus pictus) [In our study, the lacrimal gland of the Asiatic black bear was located in the dorsal-lateral corner of the eye, between the dorsal and lateral straight muscles inside the periorbital, similarly to the representatives of the family Canidae ( pictus) ,166,167. pictus) ,156. As  pictus) , this gl pictus) , the typ pictus) , Park et pictus) , and Zwi pictus) , in the  pictus)  but of s pictus) ,160,161. pictus) , and El- pictus) , showed  pictus) ,165,166  pictus) , the pre pictus) , in the -The eyeball was a spherical shape, while the periorbita was funnel/conical-shaped and the orbit was an open type;-The cornea in Asiatic black bear did not include Bowman’s membrane;-The tapetum lucidum was cellulosum, like other Canidae;-The individual orbital openings were arranged in a manner typical for Asian black bears ;-In the anterior palpebral margin of the upper eyelid, delicate and short eyelashes were observed, while in the lower eyelids they were absent, just like in Canidae;-The presence of lymphoid follicles, diffuse lymphocytes and high endothelial venules (HEV) within the eyelids as well as in Canidae and from 7–10 conjunctival folds with numerous goblet cells characteristic only to Asiatic black bear;-The marginal part of the third eyelids was very pigmented and was thick. The third eyelid was T-shaped and present were lymphoid cells that formed the subepithelial conjunctival lymphoid follicle (CALT) characterized to Canidae and Asiatic black bear;-The superficial gland of the third eyelid was a multilobar vesicular branched complex with seromucous nature and numerous lymphoid cells formed lymph nodes concentrated around the main ducts;-The lacrimal gland was very small and had a multilobar acinar branched complex structure, producing a muco-serous discharge, a small single lymphatic papule, and numerous plasma cells located in the glandular interstitium.A detailed characterization of the structure of the orbit, eyeball, eyelids and glands of the eyeball of the Asiatic black bear has not been studied so far. In the structure of the above-mentioned elements, the presence of features typical of the Asiatic black bear but also common between the Ursidae and Canidae families was found, such as:n = 1), these results should also be considered as preliminary analyses for subsequent anatomical studies in this direction, also with the use of other research methods.The current study not only broadens the knowledge of anatomy and comparative anatomy, but it can also be useful especially for veterinarians specializing in working with exotic animals and for veterinary ophthalmologists and oncologists. Nevertheless, considering the small number of animals (