Steroid-induced osteonecrosis of the femoral head (SONFH) was a refractory orthopedic hip joint disease in the young and middle-aged people, but the pathogenesis of SONFH remained unclear. We aimed to identify the potential genes and screen potential therapeutic compounds for SONFH.The microarray was obtained for blood tissue from the GEO database, and then it identifies differentially expressed genes (DEGs). The DEGs were analyzed to obtain the differences in immune cell infiltration. The gene functional enrichment analysis of SONFH was analyzed. The PPI of DEGs was identified through the STRING database, and the cluster modules and hub genes were ascertained using MCODE and CytoHubba, and the ROC curve of hub genes was analyzed, and the tissues distribution of hub genes was understood by the HPA, Bgee and BioGPS databases. The hub genes and target miRNAs and corresponding upstream lncRNAs were predicted by TargetScan, miRDB and ENCORI database. Subsequently, we used CMap, DGIdb and L1000FWD databases to identify several potential therapeutic molecular compounds for SONFH. Finally, the AutoDockTools Vina, PyMOL and Discovery Studio were employed for molecular docking analyses between compounds and hub genes.P value < 0.01 and |log2FC|> 1. Several significant GSEA enrichment analysis and biological processes and KEGG pathway associated with SONFH were identified, which were significantly related to cytoskeleton organization, nucleobase-containing compound catabolic process, NOD-like receptor signaling pathway, MAPK signaling pathway, FoxO signaling pathway, neutrophil-mediated immunity, neutrophil degranulation and neutrophil activation involved in immune response. Activated T cells CD4 memory, B cells naïve, B cells memory, T cells CD8 and T cells gamma delta might be involved in the occurrence and development of SONFH. Three cluster modules were identified in the PPI network, and eleven hub genes including FPR2, LILRB2, MNDA, CCR1, IRF8, TYROBP, TLR1, HCK, TLR8, TLR2 and CCR2 were identified by Cytohubba, which were differed in bone marrow, adipose tissue and blood, and which had good diagnostic performance in SONFH. We identified IRF8 and 10 target miRNAs that was utilized including Targetsan, miRDB and ENCORI databases and 8 corresponding upstream lncRNAs that was revealed by ENCORI database. IRF8 was detected with consistent expression by qRT-PCR. Based on the CMap, DGIdb and L1000FWD databases, the 11 small molecular compounds that were most strongly therapeutic correlated with SONFH were estradiol, genistein, domperidone, lovastatin, myricetin, fenbufen, rosiglitazone, sirolimus, phenformin, vorinostat and vinblastine. All of 11 small molecules had good binding affinity with the IRF8 in molecular docking.The microarray dataset GSE123568 was obtained related to SONFH. There were 372 DEGs including 197 upregulated genes and 175 downregulated genes by adjusted The occurrence of SONFH was associated with a “multi-target” and “multi-pathway” pattern, especially related to immunity, and IRF8 and its noncoding RNA were closely related to the development of SONFH. The CMap, DGIdb and L1000FWD databases could be effectively used in a systematic manner to predict potential drugs for the prevention and treatment of SONFH. However, additional clinical and experimental research is warranted. Osteonecrosis of the femoral head (ONFH) was a multifactorial refractory orthopedic hip joint disease in the young- and middle-aged people younger than 50 years, in which clinical manifestations appeared as the prolonged pain in the hip or groin area, occasional knee pain and limited hip rotation movement , 2. In CTo data, an increasing number of studies had shown that immune cell infiltration played a significant role in the initiation, progression and metastasis of SONFH. CIBERSORT, an analysis tool that used high-throughput transcriptomic data to evaluate the expression of immune cells and obtained various immune cell proportions from samples, had been widely employed in a variety of diseases, such as osteoarthritis , lupus nThe National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database , a databhttps://www.ncbi.nlm.nih.gov/geo/) database in SONFH and non-SONFH. The following keywords were used as a screening criterion: (1) “Steroid induced Osteonecrosis of the Femoral Head” or “SONFH”; (2) Homo sapiens; and (3) “Expression Profiling by array” or “Expression profiling by high throughput sequencing.” Finally, only one datasets GSE123568 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123568), which was submitted on Dec 10, 2018 and updated on Jan 01, 2020 by Zhang et al. and included 10 non-SONFH (following steroid administration) samples and 30 SONFH samples. The platform of the dataset was GPL15207 [PrimeView] Affymetrix Human Gene Expression Array. The raw data of GSE123568 were downloaded as CEL files, and the platform files were downloaded as TXT files.The microarray dataset was used to obtained for blood tissue from NCBI GEO (We used the robust multiarray average (RMA) method based on the “affy” R package to pre-process and normalize the original files. The high-dimensional sequencing data were projected into two-dimensional space for principal component analysis (PCA) to observe the overall distribution among groups of samples and identify the presence of singular samples. We then computed and visualized the PCA using “PCA” and “ggplot2” R package.2 (fold change)|> 1 and adjusted P value < 0.01. To better visualize the DEGs, the heatmaps and volcano plots are used by the “pheatmap” and “ggplot2” R package.We used “limma” R package to analysis the DEGs. The screening criteria were |logP < 0.05. Then the “ggplot2” R package was used to perform PCA clustering analysis on immune cell infiltration matrix data to draw a two-dimensional PCA map. The “corrplot” R package was used to draw a correlation heatmap to visualize the correlation of 22 types of infiltrating immune cells, and the “ggplot2” package was used to draw violin diagrams to visualize the differences in immune cell infiltration  gene sets with the “GSEA” and “clusterProfiler” R package. We set the parameter of require fields as follows: expression dataset as no collapse, number of permutations as 1000 and permutation type as phenotype. The significance threshold was set at a false discovery rate (FDR) < 0.25 and adjusted P value < 0.05.Gene set enrichment analysis , cellular component (CC) and molecular function (MF), whereas KEEG enrichment analyses demonstrated the significance of potential pathways associated with the DEGs.In addition, to analyze the identified DEGs, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed and visualized using the “clusterProfiler” and “GOplot” R package. The criterion for significantly enriched terms was set as adjusted https://string-db.org/). The cutoff criterion of a combined score was set to medium confidence ≥ 0.4, and the isolated nodes were discarded. Immediately after, the PPI network was constructed and visualized using the Cytoscape software .To gain more insight into the in-depth relationships among the DEGs, we predicted and constructed the PPI network of DEGs using the Search Tool for the Retrieval of Interacting Genes  and maximum neighborhood component (MNC). The final hub genes were identified as the intersection of top 20 hub genes calculated by the each algorithm.Concurrently, we analyzed the each clustered using molecular complex detection (MCODE), a plug-in of Cytoscape. The higher the module scores, the more the number of close interactions and enrichment it played, and the cutoff criterion was set as the k-core ≥ 5. Furthermore, the GO and KEGG pathway enrichment analyses of DEGs in the most significantly clustered modules were carried out by the “clusterProfiler” R package.The final specifically expressed hub genes expression profiles of non-SONFH and SONFH samples were analyzed, and the ROC curve was drawn using “pROC” R package. The area under the curve (AUC) could represent the intrinsic effectiveness of diagnostic tests, which was an indicator combining sensitivity and specificity between 0.5 and 1. The closer the AUC was to 1, the better the diagnosis was. The AUC had low accuracy at 0.5–0.7, moderate accuracy at 0.7–0.9 and high accuracy above 0.9.http://www.proteinatlas.org/), Bgee (https://bgee.org/) and BioGPS (http://biogps.org/#goto=welcome) databases were used to analyze the tissue distribution in human bone marrow, adipose tissues and blood. The purpose of the HPA database was to map all the human proteins in cells, tissues and organs using an integration of various omics technologies, including antibody-based imaging, mass spectrometry-based proteomics, transcriptomics and systems biology, which was funded by the Knut & Alice Wallenberg foundation. The Bgee was a database for retrieval and comparison of gene expression patterns across multiple animal species, which provided an intuitive answer to the question "where is a gene expressed," and supported research in cancer and agriculture as well as evolutionary biology. The BioGPS database was a free extensible and customizable gene annotation portal and a complete resource for learning about gene and protein function.To understand the specific expression of these hub genes, the Human Protein Atlas project , miRDB (http://mirdb.org/) and ENCORI , were used to predict target miRNAs of hub genes, and the final miRNAs were selected at least three databases. Next, we used Cytoscape software to construct the mRNA-miRNA network based on the relationship between miRNA and target mRNAs.The three online miRNA databases, including TargetScan . We used ENCORI to predict lncRNAs by the tools of mRNA-ceRNA network. The parameter of the mRNA-ceRNA network was set as the followed screening criteria: mammalian, human genome, h19 assembly, miRNA number ≥ 2, 2 was immediately frozen in liquid nitrogen, and RNA was extracted according to the protocol. The mRNA transcripts were quantified by qRT-PCR using Thermo Scientific™ NanoDrop Lite and a CFX96 Touch Real-Time PCR Detection System .A total of 14 cartilage samples  were collected from Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The Ethical Committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine approved this study, and the respective patient provided informed consent in a written form. At artificial hip replacement, samples of femoral neck fracture and SONFH were collected, the cartilage of 1*1 cm−ΔΔCt method. The sequences of primers used in this study are listed in Table The amplification conditions were set as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 60 °C for 30 s. Then, a melting curve was established to obtain the experimental data. GAPDH was used as the reference gene, and all qRT-PCRs were conducted in triplicates. A standard comparative method (ΔΔCt) was used to evaluate the expression stability of the potential candidate genes. Relative target gene expression levels were calculated using the 2The potentially therapeutic drugs for SONFH were predicted using the L1000FWD, DGIdb and CMap, and the final potential drugs were selected at least three databases.The L1000FWD database recorded interactive visualization of gene expression signatures of upregulated and downregulated genes induced over 16,000 drugs and small molecules in cancer cell lines. If the lists of genes we uploaded coincided the ones stored in the database, then the drugs or small molecules mapping the lists of these genes considered similar; if not consistent, it was thought to be opposite. Therefore, we could predict small molecules by comparing identified DEGs between non-SONFH and SONFH samples, and the cutoff criterion for screening drugs and small molecules was set as similarity score < − 0.03.The DGIdb database was a web-based data visualization application that revealed various types of information related to more than 40,000 genes and 10,000 drugs, which recorded the information on interactions between drugs and their candidate gene lists. Using these sources of information in the database, we could upload the identified DEGs between non-SONFH and SONFH samples to identify their interacting drugs.The CMap database, a pioneering database of chemically induced transcriptome data on human cell lines, recorded 7000 drug-associated gene expression profiles of five cancer cell lines perturbed by 1309 compounds, which could identify a large collection of drugs and small molecule compounds that had potential therapeutic effects. The drugs with significantly negative scores that was close to − 1 were predicted as therapeutic medications for disease in the database. So, we performed an in silico drug screening for SONFH using CMap by uploading Affymetrix U133A probe IDs of upregulated and downregulated genes, and the cutoff criterion used to identify potential drug candidates was set as the CMap score ≤ − 0.65 .https://pubchem.ncbi.nlm.nih.gov/) [https://www.rcsb.org/) [−1, it indicated that the two could bind spontaneously. If the binding free energy was less than − 5 kcal mol−1, it indicated that the two had good binding affinity. The binding energy ≤ − 5.0 kcal mol−1 suggested a better affinity.The hub genes and small molecular compounds were selected for the molecular docking analysis. The sdf structure of potentially therapeutic relevant small molecular compounds was downloaded from the pubChem database (ih.gov/)  and savesb.org/) . The PyMP < 0.05 was set as the threshold for significant statistical significance.Data were presented as means (± standard deviation) or counts (percentages), and the statistical analysis of the data was performed with R software (version 4.0.2) and GraphPad Prism 9 in this study. The gene expression levels of samples in the GSE123568 and GSE74089 samples were compared using the Kruskal–Wallis test. The microarray dataset GSE123568 about identification of potential biomarkers for improving the precision of early detection of SONFH were downloaded from GEO database, which included the 30 SONFH patients and 10 non-SONFH patients (following steroid administration). In the aspect of overall design of GSE123568, the gene expression profiles were detected by microarray analysis based on SONFH patients and non-SONFH patients. Then, a list of candidate gene biomarkers of SONFH were identified by integrating differential expression data analysis and gene signal transduction network analysis. Considering that the quality of the samples was critical for subsequent analysis, the original CEL files were used to evaluate the quality of the microarray while standardizing the data. We next subjected above data to principal components analysis (PCA), which confirmed the cluster analysis data and reduced the number of input variables for the subsequent advanced statistical analysis. Samples from non-SONFH patients were clustering together and maintained a significant distance from the SONFH patients shown in Fig. P value < 0.01 and |log2FC|> 1, which included 175 upregulated genes and 197 downregulated genes in SONFH samples compared to non-SONFH samples. The DEGs were shown in the volcano plots and the hierarchical clustering heatmap by the “ggplot” and “pheatmap” R package in Fig. There were 372 DEGs extracted from GSE123568 based on the defined criteria of an adjusted The GSEA enrichment analysis was used to assess the distribution trend of a predefined set of genes in lists of degree sequencing associated with the phenotype, thus judging its contribution to the phenotype. In order to unravel whether the identified sets of genes showed statistical differences between non-SONFH and SONFH patients for the abovementioned data, GSEA was analyzed and visualized based on the c2 gene sets in the MSigDB database using “GSEA” and “clusterProfiler” R package in Fig. P value = 0.001), nucleobase-containing compound catabolic process , positive regulation of cellular component biogenesis  and leukocyte differentiation  were significantly enriched in SONFH patients.As shown in Table q-value) < 0.25 and adjusted P value < 0.05, which included 51 gene sets positively correlated with the SONFH patients, such as NOD-like receptor signaling pathway, MAPK signaling pathway, FoxO signaling pathway and insulin signaling pathway, and which included 3 gene sets positively correlated with the SONFH patients, including cell cycle, biosynthesis of cofactors and olfactory transduction.We found that 54 KEGG pathway gene sets were significantly enriched at FDR  in SONFH, the online database ENCORI 3.0 was used to predict the lncRNAs that interact with the IRF8 and 10 target miRNAs. Finally, we obtained 8 corresponding upstream lncRNAs and 10 target miRNAs and IRF8. The ceRNA regulatory network was constructed and visualized using “ggalluvial” R package in Fig. The expression levels of the IRF8 were examined by the qRT-PCR in 14 cartilage samples. IRF8 was reported to be significantly dysregulated in SONFH compared with healthy samples, indicating that the results were reproducible and reliable, as shown in Fig. Using the L1000FWD, 2298 drugs with a similarity score < − 0.03 were screened by a reverse screen of the 372 DEGs in the database. We then analyzed and found 1284 drugs or molecular compounds that could reverse the expression of the 372 DEGs using the DGIdb database. Furthermore, 760 small molecules that could be associated with development of SONFH were identified by the CMap database using the 372 DEGs based on an average score < − 0.65. As shown in Fig. There were only IRF8 that was identified by CytoHubba and involved in the ceRNA regulatory network and 11 therapeutic small molecules including sirolimus, lovastatin, myricetin, rosiglitazone, vinblastine, genistein, estradiol, domperidone, phenformin, vorinostat and fenbufen in the intersection of CMap, DGIdb and L1000FWD databases. We carried out molecular docking analysis of 11 small molecules and IRF8. The smaller the binding free energy, the more stable the conformation formed by the ligand and the receptor. The results of binding energy between core target proteins and the key active ingredients are shown in Table SONFH referred to the series of pathological changes or interruption under the application of glucocorticoid, to the placement of bone marrow components and bone cell death, and subsequent autologous repair leaded to structural changes in femoral head structure and clinical manifestations until collapse . DespiteP value < 0.01 and |log2FC|> 1. The pathway enriched by GSEA of all genes mainly involved regulation of cytoskeleton organization, nucleobase-containing compound catabolic process, positive regulation of cellular component biogenesis and leukocyte differentiation., which provided some evidence that the most significant enriched gene sets that were correlated with SONFH patient were related to metabolism. In addition, the GO and KEGG enrichment analysis of DEGs were mainly related toneutrophil activation, neutrophil-mediated immunity, neutrophil degranulation, neutrophil activation involved in immune response, cytokine secretion, myeloid cell differentiation, positive regulation of cytokine production, osteoclast differentiation and viral protein interaction with cytokine and cytokine receptor. Cytokines were bioactive small glycoproteins, which could act as cell-to-cell signaling molecules that mediate interactions between immune cells and participate in the inflammatory response [We selected and downloaded the GSE123568 dataset from the GEO database and identified a total of 372 DEGs including 175 upregulated genes and 197 downregulated genes in SONFH samples compared to non-SONFH (following steroid administration) Samples based on the defined criteria of an adjusted response . It is wresponse . The aboresponse . The aboWe performed a comprehensive evaluation of SONFH immune infiltration with the CIBERSORT software to further explore the role of immune cell infiltration in SONFH. We found that activated T cells CD4 memory, T cells gamma delta, T cells CD8, eosinophils, B cells naïve, B cells memory, neutrophils, activated NK cells and resting mast cells might be related to the occurrence and development of SONFH. Bradley and his colleagues’ studies showed that the inflammatory infiltrate in SONFH patients comprised primarily CD4(+) T cells and CD68(+) macrophages, the latter of which expressed increased levels of cellular adhesion molecules . Tianbo Thereafter, the PPI network of these DEGs in SONFH was constructed by using the STRING database, and the node topological features and internode interactions were analyzed to reveal the biological mechanisms associated with the PPI network by using the NetworkAnalyzer plug-in in Cytoscape. The biological networks of the organism with stable balance might be composed of several functional modules, whose complex modules and interactions often lead to the same biological processes, and which provided a new idea for the biological functions that constitute the various components of the complex network. A total of 3 cluster modules from the PPI network were extracted by MCODE analysis based on k-core > 5. Cluster 1, which comprised 19 nodes and 88 edges, showed the highest cluster score (K-core: 9.778); this cluster was all upregulated DEGs including PINK1, BCL2L1, BNIP3L, MAP1LC3B, GABARAPL2, CASP1, PTEN, FAS, BID and FOXO3. The final filtered hub nodes also varied depending on the filtering criteria. These were 11 hub genes involved in SONFH were further identified by intersecting the results within at least three algorithms from the four algorithms of cytohubba including MCC, MNC, DMNC and degree; these genes included FPR2, LILRB2, MNDA, CCR1, IRF8, TYROBP, TLR1, HCK, TLR8, TLR2 and CCR2. All of these hub genes identified from CytoHubba were present within the 3 cluster module results by MCODE in Cytoscape. Eleven hub genes were all upregulated in the SONFH patients, and ROC curve analysis suggested that these genes had higher diagnostic value in combined with their expression levels in the SONFH and non-SONFH samples. Overall, these eleven hub genes played a crucial role in the molecular level of immune response-regulating signaling pathway, inflammatory response, activation of macrophage, cellular response to cytokine stimulus and phagocytosis. The specific expression level of all 11 hub genes was of highest available score in blood compared to bone marrow and adipose tissue in the Bgee and BioGPS databases which provided us with some novel potential therapeutic targets for SONFH.In addition, we constructed an mRNA-miRNA co-expression network and ceRNA networks to elucidate the pathogenesis of SONFH at the transcriptome level. In this study, only one hub gene in the miRNA-gene network, which were predicted at least Targetsan, miRDB and ENCORI databases, was IRF8. Therefore, we speculated that IRF8 might play an important role in the occurrence and progression of SONFH. Evidence from previous studies in animals and humans showed that IRF8, a transcription factor important for myelopoiesis and osteoclastogenesis, established cell type-specific enhancer landscapes in osteoclast precursors and mature osteoclasts in the orthopaedic disease . We specMiRNAs was an endogenous noncoding RNA molecule of length 20-24nt, which could regulate gene expression and repress translation at the post-transcriptional level of target genes by targeting the 3′UTR region of the mRNA , 47. HowTo predict the potential effective therapy for SONFH, we applied the CMap, DGIdb and L1000FWD databases to determine 11 therapeutic small molecular compounds that might reverse the abnormally high expression of the 372 SONFH-related DEGs, including vorinostat (HDAC inhibitor), estradiol (contraceptive agent), fenbufen (cyclooxygenase inhibitor), phenformin (AMPK activator), rosiglitazone (insulin sensitizer), genistein (tyrosine kinase inhibitor), domperidone (dopamine receptor antagonist), vinblastine (microtubule inhibitor), myricetin (androgen receptor agonist), lovastatin (HMGCR inhibitor) and sirolimus (MTOR inhibitor). Mounting epidemiological studies confirmed that using these several databases, such as CMap, DGIdb and L1000FWD, was a good strategy and method for conducting drug repositioning research . Xu et aAlthough the above approaches elucidate an association between IRF8 and the SONFH and explore the "multi-targets and multi-pathway" patterns in the development and progression of SONFH, our approach still has some shortcomings, such as the reliance on the reliability of the GEO database and variable data quality, which can substantially affect the analysis of gene expression in the results. Further experimental and clinical research on drug efficacy in vitro cell with different level of IRF8 expression and these related noncoding and potential therapeutic molecules is urgently needed to demonstrate their functions.In this present study, a comprehensive bioinformatics approach based on the GSE123568-assisted analysis of expression changes in key genes revealed that in SONFH patients compared to non-SONFH patients, we found that 372 DEG co-overlapping, including 197 downregulated DEG and 175 upregulated DEG and that activated T cells CD4 memory, B cells naïve, B cells memory, T cells CD8 and T cells gamma delta might be involved in the occurrence and development of SONFH. At the same time, we identified IRF8,10 target miRNAs, 8 corresponding upstream lncRNAs and 11 potentially drugs, which provided insight into the pathogenesis and treatment of SONFH.


IQSEC2 gene cause X-linked intellectual developmental disorder-1 (XLID1), characterized by a variable phenotype including developmental delay, intellectual disability, epilepsy, hypotonia, autism, microcephaly and stereotypies. It affects both males and females typically through loss of function in males and haploinsufficiency in heterozygous females. Females are generally less affected than males. Two novel unrelated cases, one male and one female, with de novo IQSEC2 variants were detected by trio-based whole exome sequencing. The female case had a previously undescribed frameshift mutation , and the male showed an intronic variant in intron 6, with a previously unknown effect (NM_001111125:c.2459+21C>T). IQSEC2 gene expression study revealed that this intronic variant created an alternative donor splicing site and an aberrant product, with the inclusion of 19bp, confirming the pathogenic effect of the intron variant. Moreover, a strong reduction in the expression of the long, but also the short IQSEC2 isoforms, was detected in the male correlating with a more severe phenotype, while the female case showed no decreased expression of the short isoform, and milder effects of the disease. This suggests that the abnormal expression levels of the different IQSEC2 transcripts could be implicated in the severity of disease manifestations.Pathogenic hemizygous or heterozygous mutations in the  IQSEC2), abundantly expressed in the brain which plays an important role in neuronal development, excitatory synapses and synaptic plasticity [IQSEC2 gene cause X-linked intellectual developmental disorder-1, XLID1, (MIM: #309530), preferentially characterized by moderate to profound intellectual disability [IQSEC2 mutations affect both sexes, females are variably and generally less affected than males [The IQ motif and SEC7 domain containing protein 2 is coded by an X-chromosome gene  in human females. As a consequence, females carrying a pathogenic mutation in the X chromosome do not manifest or have milder symptoms of the disease while males show the affected phenotype. However, isorders ,9.IQSEC2 gene, resulting from alternative promoter usage and splicing. The largest isoform  is 6037 bp in length and includes 15 exons which encode for a 1488 amino acid (aa) protein that is regulated by the upstream promoter (P2) and it is the most prominent isoform, expressed predominantly in the brain. An alternative downstream promoter (P1) regulates at least two other isoforms. The shortest isoform  includes three alternative exons, which produce a 922 bp mRNA encoding a 73 aa protein. Thus, this isoform has a different coding region with distinct 5′UTR, N- and C-termini compared to the large isoform. Even though very little is yet known about this short protein, although its highly conserved sequence suggests an important role. In addition, another intermediate isoform , which undergoes alternative splicing, combines exons of both the short and large isoforms, producing an intermediate-sized transcript of 5208 bp that is translated into a 949 aa protein [Different isoforms have been described for the IQSEC2 encodes the IQSEC2 protein, also known as BRAG1 (brefeldin A resistant Arf-guanine nucleotide exchange factor 1), a guanine nucleotide exchange factor (GEF) present in the postsynaptic density of synapses (PSD) in the forebrain. The principal substrate of IQSEC2 is the ADP-ribosylating factor 6 (ARF6) which regulates actin dynamics and membrane trafficking in neurons. [neurons. . BRAG1 ineurons. . Relatedneurons. , N-termineurons. . While thttps://www.udninternational.org/, accessed on 28 July 2022) [The Spanish Undiagnosed Rare Diseases Program, SpainUDP, aims to find a diagnosis for patients with unsolved rare diseases by applying genomic analysis together with deep phenotyping  and is aly 2022) . Here, wThe ND0673 male patient exhibits a severe neurological phenotype including psychomotor delay and hypotonia, detected at approximately 8 months of age, with epileptic spasms appearing at 5 years of age. Simultaneously, to the onset of epileptic encephalopathy, intellectual disability and autistic traits, he suffered severe neurologic regression, including a decrease in motor skills with ataxia, spasticity and gait difficulties. The ND1443 female patient has a less severe phenotype, with a neonatal normal period followed by global developmental delay. Currently, she has a neurodevelopmental disorder with behavioral problems, autistic features and language impairment.Description of the phenotype of both cases is included in Exome sequencing of both families (ND067 and ND144) identified two potentially damaging loss of function variants in the IQSEC2 gene occurring de novo in probands. None of these variants had been previously reported in either ClinVar or gnomAD databases. The constraint score provided by the gnomAD based on the ratio of the observed/expected (o/e) number of loss of function variants in a gene, which shows for the IQSEC2 gene, a very low o/e ratio of 0.03 with a CI (0.01–0.13). This indicates that IQSEC2 has a high probability of being loss of function intolerant.The male patient ND0673 was found to have an intronic variant at the genomic coordinate ChrX:g.53277882G>A (GRCh37), which corresponds to a hemizygous substitution C>T at the position +21 within intron 6 (NM_001111125:c.2459+21C>T). We suspected that this variant might interfere with the proper slicing of intron 6. The amplification of IQSEC2 mRNA with primers spanning exons 6–7 A showed The female patient ND1443, was found to have a heterozygous de novo variant consisting on a single adenine insertion, ChrX:g.53265654dup (GRCh37), located in exon 13 (NM_001111125:c.3300dup). The mutation produces a frameshift leading to the early termination of protein translation (p.Leu1027Profs*79).To determine the impact of the identified variants in the expression of the different IQSEC2 isoforms, we separately quantified the level of long isoforms (NM_001111125) and the short isoform (NM_001243197). For the analysis of the long isoforms expression, primers in two different regions were used, one at the proximal part (exons 2–3) and another one at a more terminal region (exons 12–13) A. The prIQSEC2 de novo truncating mutations were identified through whole exome sequencing in two patients from the Spanish Undiagnosed Rare Diseases Program, SpainUDP: an intronic variant in the ND0673 male patient (c.2459+21C>T) and a frameshift variant in the ND1443 female patient (p.Leu1027Profs*79), increasing the list of pathogenic mutations associated with this X-linked neurodevelopment syndrome [IQSEC2 pathogenic mutations used to be more severe in affected males than females. In accordance, the phenotype shown in our patients revealed much worse clinical manifestations in the male than in the female in terms of motor deficiency, intellectual disability and epilepsy. In this regard, previously described cases with the same mutation showed more severe developmental delay in males, providing evidence for sex differences in the severity of the disease [Two novel  disease .IQSEC2 variant (c.2459+21C>T) found in the ND0673 male patient has not been previously described. It creates a new splice donor site, producing an aberrant transcript leading to a frameshift and premature protein termination. Some other splicing variants have been described in IQSEC2, although these types of variants are less frequent than other loss of function variants [The de novo intronic variants . In our IQSEC2 remains complex. It has been suggested that the phenotype is influenced not only by the variant effect, but also by the sex of the patient and by the protein-affected domain. It is described that patients harboring de novo IQSEC2 mutations disrupting of the C-terminus of the IQSEC2 protein relate a mild phenotype [The genotype–phenotype relationship for pathogenic variants in henotype , and thahenotype . Missenshenotype ,18. In ghenotype . Truncathenotype , are genhenotype . Male pahenotype ,19,20, whenotype ,21. IQSEC2 gene is relevant for the phenotype. According to a previous study [Both the type and position of the mutation within the us study , missensus study . Despiteus study . The IQ-us study  and theiIQSEC2 variants in females have led to undetermined results in terms of phenotype severity [IQSEC2 is a gene that escapes XCI in humans [IQSEC2 expression, is globally similar in males and females, as we observed in our healthy parents [IQSEC2 expression, but the expression was higher than in the male patient, likely because of the expression of the other X chromosome. The similar expression of both the father and mother samples indicates that although the IQSEC2 gene escapes to XCI, there must be a compensatory mechanism to ensure an accurate IQSEC2 dosage. The XCI is a complex process in females, which occurs at both the tissue and cellular levels [IQSEC2 dosage probably determined by epigenetic modifications, modifier genes or skewed X inactivation which contribute to the wide clinical variability [Regarding females, the less clinically affected phenotype is influenced by the presence of a normal X chromosome . Neverthseverity  and haven humans . Some ge parents ,24,25. Tr levels . Recent r levels . Altogetiability . IQSEC2 gene by the alternative splicing of different exons and the use of an alternative promoter. Although the IQSEC2 protein is mainly expressed in the nervous system , there are differences in the expression levels of IQSEC2 transcript isoforms across tissues. The long (NM_001111125) followed by the short isoform (NM_001243197) are the two more abundantly expressed isoforms in the brain; however, the short isoform is the main isoform expressed in muscle. The implication that different levels of the isoforms might have in the disease manifestations is still unknown and should be explored in more detail. Our quantitative analysis of the expression in the whole blood of the long isoform and the shortest isoform in the two patients reveal differences in the male and female patients. In addition to the expected decrease in the long IQSEC2 isoform, with a more pronounced reduction in the male than in female, we almost found an absence of expression of the shortest isoform in the male but not in the female. This suggests that the intronic variant found in patient has an impact in the expression of the short isoform. The mechanism by which the variant affects the expression of this isoform is unknown. A potential mechanism that would explain this finding is that variant c.2459+21C>T interferes with the biding of a transcription factor. The preliminary analysis of neuronal transcription factors whose binding might be affected by the variant shows that the wild type sequence is a predicted binding site for the transcription factor ASCL1 that would be disrupted by the presence of the variant. Interestingly, the c.2459+21C>T variant would introduce a binding site for neural transcription factor SP9  and one female ND1443), were recruited by the SpainUDP [43, were Genomic DNA was extracted using Qiagen QIAamp DNA kit. Trio-based Whole Exome Sequencing (WES) libraries were prepared using Nimblegen MedExome + ChrMit using HS2000 v4, 2x100 bp, in the ND067 family and Nextera Flex Enrichment kit (Illumina), and sequencing was carried out in a NextSeq 500 Illumina sequencer using High Output Flow Cell 2x75 cartridges  in the ND144 family. The DNA quality and concentration of the pre- and post-enriched libraries were assessed using the Agilent Technology 2100 Bioanalyzer and the Quantifluor system . The quality of raw output reads was first assessed using FastQC software v.0.11.3. Next, low-quality bases were filtered and trimmed with Trimmomatic v.0.33. This tool scans sequence reads and removes the 3′ ends where the average quality of bases within the specified window size falls below the selected quality threshold. We defined a sliding window size of 4 and an average quality of 15. Reads shorter than 70 bases in length were excluded from further analyses. A minimum of 86% of target exome regions were sequenced at read depths of at least ×20, whilst mean read depths were consistently above ×75 in all cases. After preprocessing, sequences were aligned to the human reference genome “Human_g1k_v37” using BWA v0.7.12, and PCR duplicate reads were marked with Picard v1.140. Local realignment of reads, quality recalibration and variant calling were jointly performed for each family trio using the Genome Analysis Toolkit HaplotypeCaller (GATK v3.4), in line with GATK best practices ,27. KGGSIQSEC2 expression analysis approaches were designed to evaluate the effect of the intronic variant found in the male case on one hand. On the other hand, we quantitatively analyzed the large and short IQSEC2 transcript isoforms.Different IQSEC2 gene transcripts at the exons 6–7 junction (IQSEC2_Fwd: 5′GTGGGAGTGGCTCACTTCAT3′ and IQSEC2_Rv: 5′AGACTCATCGAAGCCTTCAGC3′). PCR products were subsequently loaded on a 2% agarose gel and electrophoresed to confirm the expected amplicon size (228 bp). Finally, target bands were excised, purified using QIAquick Gel Extraction Kit (Qiagen) and sequenced to check for mutation-induced effects on the splicing pattern in the patient.The total RNA from blood leukocytes of all family members was isolated using the Speedtools Total RNA extraction kit (Biotools). In addition, RNA was also extracted from muscular biopsy obtained from theND0673 male patient. Then, RNA was reverse transcribed by Maxima First Strand cDNA Synthesis kit (Thermoscientific). The total RNA input for RT-PCR was 1 µg. To check the effect of the intronic variant located in intron 6 identified in the male case (ND0673), the obtained cDNA was amplified using a pair of primers designed to span the IQSEC2 transcript isoforms in the patients relative to their parents. Commercially available Taqman probes  that could discriminate between different IQSEC2 isoforms were obtained. Probe Hs00323052_m1, which binds at the exons 2–3 junction, was used to detect the presence of the longest isoform (NM_001111125), and probe Hs06089735_m1, recognizing the alternative exons 2′-3′ boundary, was used for the shortest isoform (NM_001243197)  assays were designed and performed on both families to investigate whether the detected variants caused changes in the levels of expression of the different 1243197) A. We alsTranscription factors binding predictions from the JASPAR2022_CORE_vertebrates_non-redundant_v2 collection were analyzed using the Tomtom tool  in the M


In vitro studies using human embryonic stem cells (hESCs) are a valuable method to study aspects of embryogenesis, avoiding ethical issues when using embryonic materials and species dissimilarities. The xenograft teratoma assay is often traditionally used to establish pluripotency in putative PSC populations, but also has additional applications, including the study of tissue differentiation. The stem cell field has long sought an alternative due to various well-established issues with the in vivo technique, including significant protocol variability and animal usage. We have established a two-step culture method which combines PSC-derived embryoid bodies (EBs) with porous scaffolds to enhance their viability, prolonging the time these structures can be maintained, and therefore, permitting more complex, mature differentiation. Here, we have utilised human embryonic stem cell-derived EBs, demonstrating the formation of tissue rudiments of increasing complexity over time and the ability to manipulate their differentiation through the application of exogenous morphogens to achieve specific lineages. Crucially, these EB-derived tissues are highly reminiscent of xenograft teratoma samples derived from the same cell line. We believe this in vitro approach represents a reproducible, animal-free alternative to the teratoma assay, which can be used to study human tissue development. Understanding the events that occur during early embryogenesis and tissue differentiation continues to be a key aim in the field of human development, as well as being fundamentally important in fields such as regenerative medicine and pluripotency. Although much has been achieved and elucidated since the initial derivation of human embryonic stem cells in the 1990s ,3, and tOther techniques available to study human tissue differentiation include the xenograft teratoma assay. Perhaps under-utilised as an option to investigate human stem cell differentiation in the context of human embryonic development, the assay has long been considered the ‘gold standard’ method to confirm pluripotency in novel putative hPSCs ,9,10. ThHowever, the xenograft teratoma assay itself is not without issue. The principle of the ‘3Rs’ in animal research was originally proposed by Russell and Burch in 1959 , but witPreviously, we have shown the ability to prolong the viability and enhance the differentiation of EBs through seeding and maintenance on a porous, polystyrene membrane, resulting in the formation of recognisable tissues from the three germ layers . We prev2, 95% air. On reaching 70–80% confluency, cells were passaged via an enzyme-free method using ReLeSR  to detach and split into fresh plates at a ratio of 1:6.Human embryonic stem cell line H9  was maintained in feeder-free conditions in 6-well plates , coated with Matrigel hESC qualified matrix  and prepared and diluted according to the manufacturer’s instructions. Cells were cultured in mTESR Plus medium  and prepared according to the manufacturer’s instructions, with daily media changes and maintenance in 37 °C 5% COTM 800 plates (Stem Cell Technologies) were used to create embryoid bodies of a uniform size. Plates were prepared according to the manufacturer’s instructions prior to use, by washing in culture medium and centrifuging at a high speed to dislodge any air bubbles in the base of the microwells. Prior to EB formation, Rho kinase inhibitor (ROCK inhibitor) Y27632  was added to wells containing 70–80% confluent H9 cells for 60 min at a final concentration of 10 μM to promote single-cell survival. ROCK inhibitor was also added to media to be used during counting and EB formation at the same concentration. Following incubation in ROCK inhibitor, H9 cells were detached as described using ReLeSR, gently pipetted to form a single cell suspension and counted using the Trypan Blue exclusion assay to obtain accurate viable cell numbers. Cells were resuspended in differentiation medium, consisting of Knock Out DMEM , 20% Knock Out Serum Replacement (Fisher Scientific UK), 1 mM L-glutamine (Fisher Scientific UK), 0.1 mM non-essential amino acids (Fisher Scientific UK), 0.1 μM β-mercaptoethanol (Fisher Scientific UK) and 10 μM Y27632, and seeded at a density of 4.5 × 106 cells per well to form 300 EBs of approximately 15,000 cells. EBs were formed over 24 h by incubating the plate at 37 °C 5% CO2, 95% air, before transferring to suspension culture in sterile non-treated 6-well plates AggrewellTM and suspension culture stages.To differentiate towards the ectoderm lineage, a combination of protein and small molecules were used as outlined in the International Stem Cell Initiative Study 2018 were used see . Dorsomo® Polaris polystyrene membranes , which consist of 3 µm and are not commercially available, washed in 70% ethanol overnight, before being rinsed twice with sterile phosphate buffered saline solution (PBS). Inserts were then coated using Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning) to facilitate EB attachment before being placed in 6-well plates with 5 mL of the appropriate culture medium in the well consisting of either Knockout DMEM, 20% Knockout Serum Replacement (Fisher Scientific UK), 1 mM L-glutamine, 1% non-essential amino acids and 0.1 μM β-mercaptoethanol or high glucose DMEM (Fisher Scientific), 10% Foetal Bovine Serum , 2 mM L-glutamine, 55 µM β-mercaptoethanol, 0.1 mM nonessential amino acids and 100 U/mL Penicillin/Streptomycin (Fisher Scientific UK).Well inserts containing Alvetex® inserts, with around 50–100 EBs placed on each polystyrene insert and allowed to attach overnight at 37 °C, 5% CO2 and 95% air. The following day wells were topped up to 10 mL total media. EBs were then maintained with regular media changes for a defined timescale, before fixation in 4% paraformaldehyde  at 4 °C overnight.Once PSC-derived EBs had formed, they were transferred to the prepared AlvetexFollowing fixation, samples were washed twice in PBS and dehydrated through a series of ethanols. Samples were dehydrated in 30% and 50% ethanol before staining in 0.1% crystal violet in 70% ethanol to improve visualisation of 3D structures during processing, embedding and sectioning. Samples were then further dehydrated through 80%, 90%, 95% and 100% ethanols, before incubation in Histoclear I for 20 min , and subsequently transferred to a 50:50 solution of Histoclear I and paraffin wax (Fisher Scientific UK), with incubation at 60 °C for at least 30 min. Samples were then transferred to 100% molten paraffin wax and incubated at 60 °C for a further 60 min. Inserts were embedded in paraffin wax using plastic embedding moulds and orientated appropriately to allow for longitudinal and transverse sectioning relative to the insert. Wax blocks were sectioned at 6 µm using a Leica RM2125 RT Microtome and mounted onto SuperFrost™-charged microscope slides (Fisher Scientific UK).In order to assess structures present, slides were stained using haematoxylin and eosin (H&E). Slides were deparaffinised in Histoclear I and rehydrated through a series of ethanols, finishing in distilled water. Slides were stained in Mayer’s haematoxylin  for 5 min, before washing in distilled water for 30 s. Slides were incubated in alkaline ethanol for 30 s to blue the nuclei, before incubation in 95% ethanol for 30 s. Slides were finally stained in eosin dissolved in 95% ethanol for 1 min, before dehydration through 95% and 100% ethanols. Slides were dehydrated again in 100% ethanol and cleared in Histoclear I for 5 min each before mounting using glass coverslips and Omnimount .Slides were deparaffinised in Histoclear I for 15 min before being rehydrated through a series of ethanols, finishing in PBS. Antigen retrieval was performed by incubating the slides in citrate buffer (pH 6) for 20 min at 95 °C. Once slides had cooled, samples were incubated in blocking buffer consisting of 20% normal calf serum in 0.4% Triton-X−100 in PBS for 1 h at room temperature. Primary antibodies diluted in blocking buffer were then added to the slides at the appropriate concentrations see  and incu5 cells per 100 μL, and subsequently, 100 μL of cell suspension was combined with 100 μL of Growth Factor Reduced Matrigel matrix . This was loaded into 1 mL syringes with 21G needles which had been cooled prior to use. Loaded syringes were kept covered and maintained on ice during transport to the animal facility. The animal hosts were adult male nude (nu/nu) mice, and each host was injected subcutaneously with one loaded syringe into each flank, with three mice injected for each condition giving a potential for six total tumours per condition. Mice were examined regularly during the assay and when tumour formation was noted, the growth was measured at least once a week until a size of 1 cm2 was reached, at which point the animal was sacrificed and the tumour surgically removed. All procedures were completed under licence and permission according to the guidelines of the Home Office, United Kingdom.H9 hESCs were routinely maintained in feeder free conditions as described above. On the day the assay was commenced, cells were detached using 0.25% trypsin/2 mM EDTA (Fisher Scientific) and counted using the Trypan Blue Exclusion Assay. The cell suspension was adjusted to a density of 5 × 10Currently, the xenograft teratoma assay is the only available in vivo method to assess the developmental potency of human pluripotent stem cells, due to the inability to perform other more stringent methods which use chimeric organisms, such as germline transmission or tetraploid complementation. The teratoma assay demonstrates the ability of a hPSC line to form tissue structures from all three primary germ layers, with For many years, embryoid bodies have been a valuable model to study basic aspects of human embryogenesis and as an initial step for several differentiation protocols. Nevertheless, as the EB increases in size, differential oxygen and nutrients gradients are formed across the structure, which affects the differentiation, viability and proliferation of the PSCs and the extent to which EBs can be utilised A. We havWe have previously demonstrated how maintenance of EBs on porous membranes enhanced their viability and reduced evidence of a necrotic core . In ordeImmunostaining was performed to confirm the identity of the tissue structures observed in the 3D cultures B, using TM and suspension stages of the method in order to form tissues from the ectoderm. The morphogen combination was taken from the International Stem Cell Initiative study [To demonstrate that it was possible to direct the differentiation of tissue structures towards a given germ layer, H9 EBs were stimulated with a combination of morphogens at the Aggrewellve study  with a mSpontaneously differentiated H9 EBs maintained on porous scaffolds were capable of developing complex, differentiated structures from each germ layer, which were highly similar to the differentiated tissue structures found in teratoma xenografts formed from the same cell population B. This sHuman embryogenesis, including organogenesis and tissue differentiation, is a complex process which involves an organised series of events, including cell proliferation, division, migration and differentiation, usually through the activation of different signalling pathways in a spatial-temporal manner. Currently, a range of both in vitro and in vivo approaches are employed to investigate these processes, each with its own advantages and disadvantages regarding the methodology and information that can be acquired. Nevertheless, the formation of complex, recognisable and mature differentiated tissues from all three germ layers using hPSCs can only be achieved with the teratoma assay see . While tEmbryoid bodies have been widely used as in vitro models to study embryogenesis and tissue differentiation and to assess the cellular pluripotency of PSCs, due to their ability to recapitulate early events of embryonic development ,28,29. HTime plays a crucial role during human development, regulating the delicate balance between proliferation, gene expression and differentiation, and influencing the growth assembly of developing tissues, including their cellular composition, size and maturity . The impDirecting the differentiation of hPSCs is incredibly useful to study specific germ layers, differentiation pathways and developmental events . In an aDespite being considered the “gold standard” method for assessing the functional pluripotency of hPSCs, the teratoma assay has low consistency in both the methodology used to perform the assay and the reporting of results. This has led to the search for alternatives, as well as suggestions on how to boost the reproducibility, reliability and status of the teratoma assay, which can provide a wealth of information beyond simple pluripotency assessment ,9,11,22.We have developed a novel in vitro method to enhance the viability, differentiation and maturation of PSC derivatives into tissue structures and have explored the capabilities of this technique using human embryonic stem cell-derived EBs. We have demonstrated that the methodology has the capacity to successfully support the complex differentiation of these structures over long culture periods and can be used to form differentiated tissues of the desired germ layer. The tissue structures within the in vitro model are derived from all three primary germ layers and are highly similar to those structures found in in vivo xenograft teratomas. In this study, we have developed a reproducible, animal-free alternative to the teratoma xenograft assay, which has a number of advantages over the in vivo method, including controllability, the potential for high throughput studies and reduced costs. This novel technique can be used in a variety of applications, including the detailed study of developmental events and tissue morphogenesis and the assessment of pluripotency of novel human PSC populations.


Prioritizing sensory attributes and consumer evaluation early in breeding trials to screen for end-user preferred traits could improve adoption rates of released genotypes. In this study, a lexicon and protocol for descriptive sensory analysis (DSA) was established for sweetpotato and used to validate an instrumental texture method for which critical values for consumer preference were set. The study comprised several phases: lexicon development during a 4-day workshop; 3-day intensive panel training; follow-up virtual training, evaluation of 12 advanced genotypes and 101 additional samples from two trials in 2021 by DSA and instrumental texture analysis using TPA double compression; and DSA, instrumental texture analysis and consumer acceptability tests on 7 genotypes in on-farm trials. The established sweetpotato lexicon comprising 27 sensory attributes enabled characterization and differentiation of genotypes by sensory profiles. Significant correlation was found between sensory firmness by hand and mouth with TPA peak positive force  and positive area . D20, NAROSPOT 1, NASPOT 8, and Umbrella were the most liked genotypes in on-farm trials . An average peak positive force of 3700 gf was proposed as a minimum texture value for screening sweetpotato genotypes, since it corresponded with at least 46 % of consumers perceiving sweetpotatoes as just-about-right in firmness and a minimum overall liking of 6 on average. Combining DSA with instrumental texture analysis facilitates efficient screening of genotypes in sweetpotato breeding programs. Breeding programs have enhanced the crop by developing varieties with superior agronomic traits and resistance to several environmental stress factors, pests, diseases  to develop a lexicon and protocol for evaluation of sweetpotato by a trained descriptive sensory analysis panel and 2) to validate a high throughput instrumental texture method to establish critical values related to consumer preference for application in sensory-guided sweetpotato breeding programs.2Ethical approval for the involvement of human subjects in this study was granted by Makerere University School of Social Sciences Research Ethics Committee (MAKSSREC 12.19.364), Centre de Coopération Internationale en Recherche Agronomique pour le Développement , CIRAD, Ethics committee, and retrospectively by the Faculty of Natural and Agricultural Sciences, University of Pretoria (NAS 236/2021).2.1Sweetpotato roots of various genotypes were obtained from the ongoing International Potato Center (CIP) breeding trials in various locations in Uganda, while some were obtained from farmers in the same areas. Roots sourced from the open market usually have a high variation within them and these were avoided. Many genotypes were used for lexicon development and sensory panel training in phases 1–3  and 5 checks . Test clones are genotypes being studied for selection and potential release as new varieties while checks are released or local varieties of known agronomic performance. This multi-location trial included clones of the 12 genotypes harvested in 2020 under the same trial. In total, 61 unique samples were studied. Another set of sweetpotato roots representing 40 clones, 7 of which were among the raw material for making a new generation of genetic improvements referred to as parents , were obtained from Mwanga Diversity Panel (MDP) population planted in 2021. Descriptive sensory profiles by the trained panel and instrumental texture measures collected from these materials were used to establish relationships between sensory texture and instrumental texture parameters.Consumer sensory analysis was conducted with 106 consumers with 7 genotypes planted as part of the on-farm trials in phase 6. The genotypes included two checks, NASPOT 8 and NAROSPOT 1; three test clones, D20, NKB3, NKB105; and two local clones, Muwulu Aduduma and Umbrella. On-farm trials are part of the final steps in a breeding cycle and were designed as participatory plant breeding where farmers are involved in breeding selections.2.2Twenty-one individuals  working at the National Agricultural Research Organization (NARO) in Uganda that had completed 50 h of prior sensory training with other roots, tubers and banana crops were recruited by the Food Biosciences and Agribusiness Program for the sensory panel in 2019. Participants were informed about the objectives and provided consent during the first training session. Candidates participated in phase 1, a first workshop of lexicon development (August 2019), with 10 continuing with the training that followed in phase 2 (October 2019). Nine panelists attended phase 3, a virtual training and analyzed sweetpotato samples in the office setting (July 2020).2.2.1A draft lexicon was developed with 21 participants during a 4-day training workshop. The panel tasted 7 genotypes of contrasting sensory characteristics .Four members of the panel were trained to prepare and evaluate the samples at their individual homes using cookware available in their kitchens. Raw sweetpotato roots were delivered to the panelists’ homes in labelled paper bags. However, the samples prepared at home were variably cooked (often undercooked), perhaps because they did not have the steaming pots which the research team uses in the laboratory. As a result, there were many cases where samples could not be evaluated. Therefore, data from this group were excluded.2.2.3In April and May 2021, 13 participants attended a 7-day training workshop where they were trained on the terms in the lexicon and how to conduct sensory evaluation of steamed sweetpotato. Among them, 12 trained panelists participated in descriptive sensory profiling of 12 advanced genotypes from DDBIO advanced field trial planted in 2020. From October to early November 2021, they participated in sensory profiling of 40 genotypes from the MDP trial population, and then from late November to December, all 13 panelists evaluated 61 genotypes from the DDBIO multi-location advanced field trial planted in 2021. In February 2022, a 4-day training, attended by trained panelists and 3 new participants, was held and 12 panelists evaluated genotypes used in on-farm trials.2.2.4Sample preparation evolved throughout the panel training phases. The roots were prepared in a central kitchen. During lexicon development (phase 1), sweetpotatoes were prepared following the commonly used local method where roots are peeled, wrapped in banana leaves, and steamed for 1 h in a saucepan as detailed in From phase 2 onwards, sweetpotato roots were prepared by cutting out a 7 cm portion  of the 12 genotypes from the DDBIO advanced field trial   to 9 (like extremely). They also rated sweetness, mealiness and firmness on just-about-right scales ranging from 1 to 5 centered by just-about-right (JAR) at 3. These attributes were previously identified as the most important drivers for consumer preference of boiled or steamed sweetpotato in Uganda .2.4.2During the pilot study, samples for consumer sensory analysis were prepared according to the method outlined in section 2.2.4. However, to align with participatory plant breeding design, women identified from the local community prepared the sweetpotato in a culturally appropriate manner. Each woman was assigned a single variety. The women peeled the raw roots and placed them in water immediately to prevent browning, wrapped the roots in banana leaves and placed the bundle in saucepans matted with grass. They covered the wrapped sweetpotato roots with more banana leaves and added water to the saucepans to steam the sweetpotatoes over a three stone fireplace with burning wood. Roots were deemed ready when the loud bubbling sound of boiling water became quieter indicating reduced water levels. The sweetpotatoes were removed from the fire, unwrapped slightly and lightly pressed using fingers to confirm that they were cooked before serving.In order to overcome the limitation of number of roots, roots of different sizes were cooked. Ready to eat sweetpotato roots were divided depending on size and medium sized roots were quartered while large roots were divided into 8 portions. The portions were wrapped in aluminum foil and labelled with random codes. Respondents were presented with the cooked samples monadically to evaluate in random order.2.52.5.1All sensory data were first organized in Microsoft Excel . For descriptive sensory analysis, data from genotypes served in duplicate (sensory replicates) were used to evaluate panel reliability in SPSS version 22  by FisheData for descriptive sensory analysis at phase 3, phase 4, and phase 6 were analyzed in SPSS analysis of variance (ANOVA) models with genotypes as fixed variable and panelists as a random factor. To visualize relationships between the sensory attributes and the genotypes, principal component analysis (PCA) using mean scores for each replicate by the trained panel was run in XLSTAT  using the covariance option. Attributes that were not found to be significantly different among the varieties by ANOVA were excluded from the PCA. During phase 3, genotypes were not found to be different (p > 0.05) in caramel aroma, off -odor, floral flavor, bitter taste, and adhesiveness, hence the exclusion of these attributes from PCA. Attributes that were excluded from the PCA at phase 4 were: caramel aroma, off odor, floral flavor, bitter taste, fibrous appearance, cooked carrot flavor, crunchiness, and adhesiveness. Caramel aroma, off odour, degree of translucency, fibrous appearance, cooked carrot flavor, floral flavor, sweet taste, bitter taste, adhesiveness, and fibrousness were excluded at phase 6.2.5.2With all texture data, means for each texture parameter, specifically average peak positive force and average positive area for the first and second compressions, were calculated. When analyzing data from descriptive sensory panel and instrumental texture analysis, a panel mean for each sensory attribute evaluated by the panel was calculated per genotype including those that were served in duplicate.In phase 4, instrumental texture parameters were correlated with sensory firmness  using Pearson correlation analysis. In phase 5, a linear regression model predicting sensory firmness was developed in XLSTAT with the best model procedure using 59 clones which had complete data from the DDBIO multi-location advanced field trial planted in 2021 as the training set and the 12 genotypes of the DDBIO advanced field trial from phase 4 as the validation set. To solve for multi-collinearity due to the strong correlation between instrumental texture parameters, only peak positive force (firmness) of the first compression was included in the model. Natural logarithms of the values of the predictors (dry matter and peak force) were entered in the model to ensure heteroscedasticity of the output residuals, and RMSE values of both the calibration and validation sets were calculated. The model was validated using descriptive sensory data from the panel and instrumental texture analysis of 39 genotypes from another population, MDP, of the same season.2.5.3To conduct penalty analysis, the 5 point just-about-right scale was collapsed into 3 categories by combining the lower end points (1 and 2), and upper point scales (4 and 5) . Penalty2.5.4Several measures from the firmness JAR question were plotted against instrumental texture measures to propose minimum and maximum values corresponding to consumer liking. Linear regression was applied to establish relationships between instrumental texture measurements of genotypes and frequencies of being ‘too soft’, ‘too hard’, or ‘just-about-right’ in firmness. Plots of the frequency of these responses were plotted against instrumental texture measures of firmness (peak force 1) and toughness (positive area 1) to produce linear prediction equations in Excel.In order to select a suitable threshold, plots of overall liking and the frequency of responses to the just-about-right question on firmness were also plotted to identify the frequency associated with a minimum overall liking of 6, which corresponds to ‘like slightly’. These were used as cut-off frequencies to calculate proposed values of instrumental texture measures. The frequencies of respondents who found the sweetpotato samples ‘too soft’ or ‘just-about-right’ in firmness were entered in their respective prediction equation to calculate the values of texture firmness (peak force) and toughness (positive area). These output values were proposed as minimum values for instrumental texture parameters that would indicate sweetpotatoes of suitable firmness for Ugandan consumers.33.13.1.1The initial lexicon comprised 36 terms describing the aroma, appearance, flavor, and texture of steamed sweetpotato  and soft and moist genotypes on the right . S36, an orange-fleshed genotype, was particularly firm while NKB3 and NKB105 were moist and soft. The second component, F2, separated varieties by orange color intensity and sweetpotato flavor. Deeply orange genotypes such as Ejumula, S36, NKB3 and NKB105 appeared towards the top of the plot, and NASPOT 11 and New Kawogo which are white, and cream fleshed towards the lower side of 3.3.1The dry matter and instrumental texture parameters for the 12 genotypes in the advanced trial are shown in Table S.7 in There was good correlation between sensory firmness and parameters of instrumental texture especially peak positive force (firmness) and positive area (toughness) of the first curve . Peak po3.4A multiple linear regression model S.1 in s was dev3.5The sensory profiles developed by the trained panel of the 7 genotypes evaluated in on-farm trials are shown on the PCA, which explains 93 % of the total variation among the genotypes . Orange 3.6The mean overall liking for the different sweetpotato genotypes ranged from 5 (NKB3) to 7  . Using t3.72 = 0.96 and R2 = 0.85 respectively). Similarly, there was a strong positive relationship between instrumental firmness and overall liking of the samples (2 = 0.92), whereas the relationship between instrumentally measured firmness and respondents who found any sample ‘too hard’ was weak (R2 = 0.48). Due to the low frequency of samples perceived as ‘too hard’, it was not considered for establishing maximum levels for instrumental texture firmness.Figure S.4 shows the relationship between overall liking and responses to just-about-right question on firmness. Using these relationships, cut-off values for frequency of respondents perceiving samples to be ‘too soft’, ‘just-about-right’ firm, and ‘too hard’ corresponding with a minimum overall liking of 6 were 50 %, 46 % and 4 %, respectively. Using the prediction models, 50 % response of ‘too soft’ corresponded with an average peak force of 3435 gf (∼3400 gf) and positive area of 6426 gf.s (∼6400 gf·s). About 46 % consumers would perceive sweetpotato firmness to be ‘just-about-right’ when peak force is 3682 gf (∼3700 gf) and positive area is 6323 gf·s (∼6300 gf·s). Thus, the minimum values of peak force and positive area were set at 3700 gf and 6400 gf·s, respectively for large scale screening of sweetpotato genotypes for consumer preferred firmness.Even though the relationship between the proportion of consumers who perceived sweetpotato as being too firm and instrumental texture parameters was linear, the small range of responses made it difficult to determine the relationship at higher proportions. It was therefore difficult to make conclusions on the maximum values of instrumental texture parameters in the current study.4In the early stages of lexicon development, panelists used a Luganda term, “kiwutta” to describe the texture, taste, and flavor of some sweetpotatoes. Upon further discussions with the panel, it was identified that “kiwutta” referred to poor quality characterized by translucent appearance, extreme hardness, moisture release, crunchiness, and high sweetness and intense floral flavors. Its derivative term muwutta was used to refer to a glassy texture by consumers of boiled potato  observedThere were two iterations of lexicon developed for steamed sweetpotato with the second refining and reducing the number of descriptive terms. Comprehensive sensory characterization of sweetpotato using many descriptors is ideal but would be challenging for routine use with breeding trials because there are many samples to analyze. Fewer attributes reduce response burden on the panelist and could thus also contribute to improved panel performance . Since tThe lexicon was modified progressively as panelists were trained with exposure to sensory characteristics of different sweetpotato genotypes, like the concept of a ‘living lexicon’ as shown by The lexicon shares common terms with others such as the lexicon for boiled sweetpotato by The sensory panel was able to discriminate samples after virtual training and evaluation of samples outside the laboratory when prepared in a centralized cooking area. The different genotypes  were differentiated based on their overall sensory profiles, and the duplicates of NASPOT 11 and MDP 452 were close together indicating the good panel performance in this setting. Nonetheless, the validity of conducting descriptive sensory analysis outside the lab cannot be concluded from the current study since panel performance was not compared with the conventional method. Future studies seeking to recommend alternative spaces in which to conduct descriptive sensory analysis should compare with laboratory evaluations.One important way of integrating descriptive sensory analysis with breeding is by developing rapid instrumental methods that can help in screening potential genotypes . The peaThe linear regression model developed to predict sensory firmness using instrumental texture consistently confirmed that there was a good relationship between the two measures of sweetpotato firmness. The model had good precision but only explained 65 % of total variation among genotypes by sensory texture. Nonetheless, with this prediction the instrumental texture method developed in this study will still be useful in helping breeders to objectively screen sweetpotato genotypes by sensory firmness earlier in the breeding process.It is possible that there are other instrumental texture or biochemical measures that could explain sensory firmness which could be added to the model and increase the variation explained and improve prediction of the model. The current method was correlated with sensory firmness and future studies should develop alternative methods that could predict textural aspects of sweetpotato related to other human perceptions of sweetpotato texture such as mealiness and fibrousness. Measures that investigate the visco-elastic properties of the sweetpotato such as stress-relaxation could be useful. Instrumental texture analysis such as the method developed in this study is simple and practical for breeding programs which could be a limitation of other texture measures. The established instrumental texture analysis method should be used to complement but not replace sensory profiling by trained descriptive sensory panels in breeding programs. Its application would be particularly useful in earlier stages of breeding when there are many genotypes to screen. It may still be necessary to use descriptive sensory panels to profile genotypes later in the breeding cycle when there are fewer genotypes and enough roots.According to the consumer evaluation and penalty analysis, D20, NASPOT 8, NAROSPOT 1 and Umbrella were the most preferred varieties and NKB3 was the least preferred variety. NASPOT 8 has previously been indicated as one of the most preferred varieties based on sensory quality . D20, NKIn addition to validating an instrumental method for evaluating firmness of steamed sweetpotato, the study proposes a minimum (3700 gf) average positive peak force of the first cycle for use as a selection criterion when screening sweetpotato genotypes in breeding trials for consumer acceptance. Following this criterion, among the 12 genotypes of the DDBIO advanced field trial planted in 2020, only three test clones: D26, S36 and S47 would qualify to proceed to the next breeding stage while NKB3 and NKB105 could have been excluded from the on-farm trial that followed. Use of this proposed minimum value of peak force screening criteria proposed in this study requires validation in a study with genotypes of a wider range of firmness, especially firmer genotypes.4.1Breeding trials typically produce few roots which limits the availability of sample material for the various quality analyses conducted to inform screening of sweetpotato genotypes. Consumer testing of food products typically requires each consumer to be presented with similar size and shape of the product. Here roots of varying sizes and shapes were cooked, and the medium and large roots were divided differently in order to serve the large number of consumers. Consumer acceptability tests were also conducted with residents of one community and the preferred ranges of sweetpotato firmness could vary by region. Nonetheless, the study establishes protocols for efficient breeding based on consumer preferred texture parameters. Further work is required to develop screening criteria for appearance and flavor characteristics.5This study established a trained descriptive sensory analysis panel for sweetpotato in Uganda. A complete sensory lexicon, preparation and evaluation protocol for sweetpotato evaluation was developed. A method for instrumental texture analysis of steamed sweetpotato using TPA was validated and used to establish the lower critical value for consumer liking of sweetpotato firmness. This criterion can be used by breeders to efficiently select potential genotypes from the large sets of genotypes in the breeding pipeline.Mariam Nakitto: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Suzanne D. Johanningsmeier: Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Mukani Moyo: Methodology, Investigation, Writing – original draft, Writing – review & editing, Supervision. Christophe Bugaud: Methodology, Validation, Investigation, Writing – review & editing, Supervision. Henriette de Kock: Methodology, Writing – review & editing, Visualization, Supervision. Layal Dahdouh: Methodology, Validation, Formal analysis, Writing – review & editing, Supervision. Nelly Forestier-Chiron: Methodology, Investigation, Validation, Writing – review & editing. Julien Ricci: Investigation, Validation, Writing – review & editing. Elizabeth Khakasa: Investigation, Writing – review & editing. Reuben T. Ssali: Methodology, Resources, Writing – review & editing, Project administration. Christian Mestres: Methodology, Formal analysis, Writing – review & editing, Supervision, Project administration. Tawanda Muzhingi: Conceptualization, Methodology, Writing – original draft, Supervision, Project administration.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


PSTS was significantly greater in PAP than in AP at level C2 on postoperative day (POD) 1 (p = 0.0001). PSTS was more prominent at levels C2–4 during PODs 2–4 for both groups. In staged combined multilevel anterior–posterior complex spine surgery, PSTS is an inevitable complication. Therefore, surgeons should monitor PSTS after surgery when performing anterior–posterior complex cervical spine surgery, especially in the immediate postoperative period after PAP surgery.The influence of the sequence of surgery in the development of prevertebral soft tissue swelling (PSTS) in staged combined multilevel anterior–posterior complex spine surgery was examined. This study was conducted as a retrospective study of patients who underwent staged combined multilevel anterior–posterior complex cervical spine surgery from March 2014 to February 2021. Eighty-two patients were identified, of which fifty-seven were included in the final analysis after screening. PSTS was measured from routine serial monitoring lateral cervical radiographs prior to and after surgery for five consecutive days at each cervical level from C2 to C7 in patients who underwent anterior then posterior (AP) and posterior then anterior–posterior (PAP) surgery. The mean PSTS measurements significantly differed from the preoperative to postoperative monitoring days at all cervical levels ( Combined anterior–posterior surgery is usually performed for complex cervical spine cases with multilevel pathologies that require both anterior and posterior decompression, correction of kyphosis, restoration of instability, and attainment of sagittal balance ,2,3,4. SPrevertebral soft tissue swelling (PSTS) has been shown to cause postoperative problems, such as airway compromise and dysphagia, in anterior cervical spine surgery ,3,5,6,7.This study was conducted after obtaining institutional review board approval from our institution (IRB number 4-2019-1279). The records of patients from March 2014 to February 2021 were retrospectively reviewed. Subjects that were included comprised patients who underwent combined treatment for multilevel complex degenerative and deformity cervical spine conditions. We excluded patients who had cervical spine pathologies involving trauma, tumor, or infection. We also excluded patients who underwent anterior cervical corpectomy fusion for any cervical spine condition. In addition, only patients who underwent anterior cervical discectomy fusion (ACDF) of at least three levels from C2 to T1 were selected. We collected data on the demographic and surgical profiles of the patients.Preoperative and routine serial postoperative lateral monitoring cervical spine radiographs from postoperative days (POD) 1 to 5 were reviewed ,10,11. Tn = 37), six were excluded for either radiographs not having been acquired on particular days during the monitoring or missing measurements due to poor radiograph quality. Among the staged AP group, only 31 were included for the final analysis. Of the staged PAP patients (n = 36), 10 were excluded for the reasons above. Among the staged PAP group, only 26 were included for the final analysis  and bioactive glass–ceramic spacers . No anterior instrumentation or plate was used to avoid limiting any posterior correction of kyphosis that could be achieved with posterior surgery, except in some cases when a plate  was used according to the surgeons’ discretion at levels C7-T1 to prevent subsidence.Posterior surgery was accomplished using a posterior approach to the cervical spine. Decompression of the cord was primarily attained by multi-segmental en bloc laminectomy. Additional decompression methods were also employed, such as posterior foraminotomy, laminotomy, and dome laminoplasty. A combination of a lateral mass and pedicle screw SYNAPSETM system  or an all-pedicle screw POSEIDONTM system  with a rod construct was used for fixation. A rod connector was used as necessary to properly link the rod with the screws in the lateral mass and pedicle screw combination construct. A crosslink was usually placed at the middle of the construct for added stability. Residual kyphosis correction was established by segmental instrumentation, which also allowed for a sustained alignment and promoted fusion. The posterior fusion and decompression levels were selected on the basis of levels of compression and the preservation of sagittal balance . AutologIn AP surgery, ACDF was performed in the first surgery stage, followed by posterior decompression and fusion in the second surgery stage. For PAP surgery, posterior decompression and the application of pedicle or lateral mass screws were performed without rod assembly during the first surgery stage. Morselized lamina bone autografts were laid lateral to the screws along the facet joints. The second surgery stage involved ACDF followed by the subsequent application of rods immediately after on the same day on the posterior side, ensuring adequate lordosis and good alignment. For both AP and PAP sequences, the interval between the first and second surgery stages was 7 days . Extubatt-test and z-test, respectively. The homogeneity of three or more proportions was assessed using the chi-square test. Differences in the prevertebral soft tissue swelling measurements for each cervical level involved across the different monitoring days were identified using repeated measures analysis of variance. The intra- and interobserver reliability of radiographic values were assessed using the intraclass correlation coefficient (ICC). All statistical analyses were conducted using STATA® V12.0  software at a significance level of 0.05. A power analysis was performed based on the 31 AP and 26 PAP subjects included for analysis. The results showed that the sample sizes for each group would yield a power of 0.80 to detect an effect size of 0.758 at a two-tailed 0.05 significance level (α). Descriptive measures on the demographic and surgical profiles of the patients are reported. Comparisons between the two means and two proportions were conducted using a p = 0.4990), sex (p = 0.1820), body mass index (p = 0.8480), American Society of Anesthesiologists classification (p = 0.5770), diagnosis (p = 0.5062), smoking (p = 0.4690), or alcohol intake (p = 0.3187)  .p = 0.0070) and the total operative time during the first and second surgery stages (p = 0.0049)  .p = 0.0000)  . Good top = 0.0001) at level C2 on POD 1. Among all other levels and across all postoperative monitoring days, however, the mean PSTS amounts between PAP and AP surgery were not significantly different. Additionally, for both groups, we noted that PSTS peaked at PODs 2–4 and, at the same time, that the differences in PSTS with respect to the preoperative measurements were more remarkable at levels C2–4, compared with the lower cervical levels  than in AP (8.53 ± 4.30 mm) surgery (l levels . None ofFactors to consider when deciding to perform combined anterior–posterior surgery for complex cervical spine cases, especially in degenerative and deformity cases, include the extent of compression, levels of congenital canal stenosis, patient symptoms, spinal alignment, compressive pathology, and the experience and surgical preference of the surgeon ,15. MostPersistent dysphagia in one patient was probably due to excess anterior and posterior segmental lordotic correction, which could cause stretching of the esophagus coupled with compression of the posterior pharyngeal wall by the vertebra ,18. AirwThe PSTS amounts differed from the preoperative to postoperative monitoring days at all cervical levels in both PAP and AP surgical groups. Similar to the findings of Suk et al., the swelling was more remarkable at PODs 2–4, and notably, PSTS was greater at levels C2–4 . This waPulmonary complications that occur after surgery include atelectasis, pneumonia, bronchitis, bronchospasm, acute respiratory failure, and pulmonary thrombosis. The incidence rates vary from 20 to 69% for atelectasis and 9 to 40% for pneumonia. The total incidence rate varies widely at 10–80% . The occThe study was limited for being retrospective and could have been made more homogenous if the surgical procedures in both groups were made uniform. In the single-center evaluation, only 57 patients were included. A nonrandom assignment of patients to each surgical group was performed at the discretion of the primary surgeon. It can still be concluded, however, that PSTS in this group of patients was significantly related to the sequence of surgery and that it was greater in PAP than in AP surgery in the immediate postoperative period in the upper airway.


The peripheral nerve sheath tumors are relatively common neoplasms, belong to the soft tissue sarcomas group, and are poorly investigated in veterinary medicine; the diagnosis is complex, and therapeutic options are limited. The platelet-derived growth factor receptors, namely the β subunit, are an important class of tyrosine kinase receptors that can be activated by genetic alterations and contribute to the process of carcinogenesis, so the inhibition of this receptor is an important therapeutic target. Using the immunohistochemical technique, this study aims to evaluate the expression of this receptor in 19 samples, 10 malignant and 9 benign tumors. The results showed that the majority of benign tumors, about 67% of cases, expressed the receptor in less than 25% of neoplastic cells and, in 80% cases of malignant tumors, the receptor was expressed in more than 25% of neoplastic cells. It was also found that, in the larger tumors, the expression of this receptor was significantly higher. With these findings it seems reasonable to speculate that the drugs able to inhibit this receptor, such as toceranib, may be considered in the therapeutic approach of these tumors.As in humans, the prevalence of tumors in companion animals is increasing dramatically and there is a strong need for research on new pharmacological agents particularly for the treatment of those tumors that are resistant to conventional chemotherapy agents such as soft tissue sarcomas (STS). Because malignant (MPNST) and benign peripheral nerve sheath tumors (BPNST) are relatively common STS in dogs, the aim of this retrospective study was to evaluate the immunohistochemical (IHC) expression of PDGFR-β, contributing to its characterization as a potential target for their treatment. A total of 19 samples were included, 9 histologically classified as benign and the other 10 as malignant. The results showed diffuse immunoexpression in the cytoplasm of neoplastic cells. Six (66.7%) BPNST expressed the receptor in less than 25% of neoplastic cells and only three (33.3%) exhibited labelling in more than 25% of neoplastic cells. In contrast, all MPNST expressed PDGFR-β, and in 8 (80%) of these samples, the receptor was expressed in more than 25% of neoplastic cells, and only 2 (20%) cases expressed the receptor in less than 25% of neoplastic cells. PDGFR-β expression was significantly higher in MPNST and larger tumors, suggesting that drugs able to inhibit the activity of this tyrosine kinase receptor, such as toceranib, may be considered in the approach of unresectable tumors and/or in the context of adjuvant or neoadjuvant therapies. Soft tissue sarcomas (STS) are tumors derived from tissues of mesenchymal origin, meaning they can arise from almost any anatomical site ,2,3,4. TMalignant peripheral nerve sheath tumors (MPNST) are a subtype of STS arising from Schwann cells, perineural cells or endoneural and perineural fibroblasts and they account for 27% of all peripheral nervous system tumors in dogs ,7. They Platelet-derived growth factor receptors (PDGFRs) α and β are tyrosine kinase type receptors (TKR) that participate in cellular physiological and pathological signaling pathways, mainly through paracrine mechanisms. In the adult physiological processes, they stimulate fibroblast and endothelial cell proliferation and are involved in tissue regeneration and fibrosis ,14. ThesHowever, PDGFRs can be aberrantly activated by several genetic alterations contributing to carcinogenesis ,20. NeopIn human medicine, PNST have been found to express specifically PDGFR-β so therapies targeting this receptor may have therapeutic potential ,24. In vTo the best of our knowledge, PDGFR-β has not been studied in canine PNST, and therefore, the goal of this preliminary study was to evaluate the PDGFR-β expression in these skin tumors through an immunohistochemical (IHC) assay, aiming to pave the way to investigate new potential therapeutic targets for effective management of patients affected by these tumors.This study was conducted using archived samples from the Veterinary Pathology Laboratory of the University of Porto, after approval by the Ethical Committee for the Animal Wellbeing (ORBEA/P314/2019). Nineteen archived formalin-fixed paraffin-embedded (FFPE), Hematoxylin and Eosin (HE)-stained canine PNST tissue samples were selected, including 10 malignant cases  and 9 benign schwannomas. All cases met the current diagnostic criteria ,34,35,36TM; Novocastra Laboratories, Newcastle, UK), according to the manufacturer’ instructions. Sections were dewaxed in xylene and rehydrated through graded alcohols. The antigen retrieval was performed using microwave radiation with Extran® solution (at 1:50 dilution) for approximately 10 min. Then, slides were cooled and washed in Tris-buffered saline . Endogenous peroxidase was blocked with the addition of Peroxidase Block solution (Novocastra™) for 12 min at room temperature. To inhibit the binding of non-specific proteins, the Protein Block solution (Novocastra ™) was added for 1 h, at room temperature. Slides were then incubated overnight at 4 °C with a rabbit monoclonal anti-PDGFR-β antibody , diluted 1:50 in TBS with 5% bovine serum albumin (BSA). After washing the slides with TBS, the Post Primary solution (Novocastra™) was added for 30 min at room temperature, followed by the addition of the Polymer solution (Novolink™) for another 30 min. After washing with TBS, slides were incubated with DAB Substrate Buffer (Novolink™) and DAB Chromogen (Novocastra™). Finally, they were washed for 10 min and contrasted with hematoxylin. Positive controls consisted of sections from canine hemangiosarcoma and the immunostaining of macrophages, fibroblasts and vascular smooth muscle cells were used as internal positive controls [Two µm thick tissue sections were subjected to IHC technique using a polymer-based system  according to a previously described method ,38. The The Ki-67 IHC method was performed as previously described by Teixeira et al. (2016)  using a Slides were examined independently by three observers  and, in case of disagreement, a consensual result was reached through simultaneous evaluation of the slide using a multi-head microscope.The results were analyzed with the SPSS software . For statistical analysis, tumors were grouped according to a cut-off value of 25% PDGFR-β positive cells. The relationship between PDGFR-β expression and clinical data, such as gender, tumor location (trunk or limbs), size (<3 cm and ≥3 cm) and breed , as well as with histopathological factors, such as tumor type (BPNST vs. MPNST) and proliferation index (Ki-67 < 20% or ≥20%) were analyzed by Fisher’s exact test. ANOVA analysis was used to compare the mean Ki-67 index with the type of neoplasia and the expression of PDGFR-β. Values of p < 0.05 were considered statistically significant.Data regarding breed, age, gender, type of tumor, tumor location, dimensions, Ki-67 proliferation index and % of PDGFR-β expression are summarized in The mean age ± SD of the animals with benign neoplasms was 10.67 ± 2.35 years while those with malignant neoplasms were 9.00 ± 3.23 years old. In this population, the PNST occurrence was similar between males (10 cases—52.6%), and females (9 cases—47.4%), although the latter presented a higher number of malignant neoplasms (6/9) when compared to males (4/10).Most of PNST was found in larger breed animals (11/14), and the majority of them were malignant . All smaTumor behavior was independent of tumor location, but a significant association was found between MPNST and higher tumor sizes ; all MPNThe PDGFR-β staining was diffuse cytoplasmatic in both benign and malignant PNST. The labelling intensity of BPNST was weak in 8 cases and intense in 1 case; in MPNST, it was weak in 6 cases and intense in 4 cases a,b. In aPDGFR-β was expressed in less than 25% of tumor cells in 6 of 9 BPNST samples (66.7%), including 1 case without immunoreactivity. All MPNST expressed PDGFR-β, with 80% of them (8/10) in >25% of cells. PDGFR-β expression was significantly higher in MPNST when compared to BPNST . The tumThe activation of PDGFRs has an important role in the carcinogenesis of some human and animal neoplasms ,20. In dDespite the small sample in this preliminary study, it was possible to verify that the age of development of PNST does not appear to be related to their biological behavior, which is in accordance with the existing literature . It has BPNST showed a significant lower expression of PDGFR-β compared to MPNST, suggesting that the receptor may have a role in the malignant transformation of these neoplasms, as also has been demonstrated in human PNST . It has Larger tumors exhibited a higher level of expression of this TKR, suggesting that it may also contribute to tumor growth and perhaps a worse prognosis, but survival studies are needed to confirm this hypothesis. In addition, malignant PNST showed a more intense PDGFR-β expression at the tumor invasive front and areas of higher cytological atypia, suggesting that it may contribute to the tumor local invasive capacities ,16,17,18®), may have a role in the treatment of the more aggressive PNST. In fact, the blockage of PDGFR-β expression with imatinib, a TKI used in human medicine, decreases the epithelial-mesenchymal transition (EMT) and the transcription of factor Slug, and it dramatically reduces the progression of human sarcomas [Considering the overexpression of PDGFR-β in MPNST and in larger tumors, it seems reasonable to speculate that tyrosine kinase inhibitors (TKI) such as toceranib (Palladiasarcomas .This preliminary investigation had the strong limitation of the low number of cases, which limits the statistical power of the results, and therefore, further studies with a large number of PNST samples are warranted. Although PNST are not very common, they may have different clinical behaviors, and their proper diagnosis should be emphasized. The diagnostic process includes a panel of immunohistochemical markers that are expensive to perform and, because of that, many of these tumors are diagnosed only as cutaneous sarcomas and not distinguished from other soft tissue sarcomas, which may have consequences in terms of prognosis and therapeutic approach.In summary, this study described, for the first time, the PDGFR-β expression in canine PNST, evidencing its presence in the cytoplasm of neoplastic cells. Despite the small number of samples, it was demonstrated that malignant and larger PNST have a significantly higher level of this TKR, suggesting that the receptor may have a role in the pathogenesis and progression of these neoplasms. These preliminary results support the need for conducting more investigations and survival studies to understand the value of PDGFR-β at both prognostic and therapeutic levels. Considering that some of these tumors may be difficult to totally excise, they may require adjuvant or neoadjuvant treatments, justifying the need for new therapeutic strategies, such as toceranib, a veterinary TKI able to target PDGFR-β.