Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA binding proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68  in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically loaded focal adhesions by modulation of integrin signaling and local delivery of β-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks. Maintenance of tissue homeostasis requires reciprocal interactions between the extracellular matrix (ECM) and cells that organize their microenvironment. Endothelial cells play a central role in tissue homeostasis and they actively contribute to growth during development, tissue patterning, and regeneration processes in which the remodeling of blood vessel networks is highly dynamic . Fine tuSAM68 (Src associated in mitosis) is an RBP present in endothelial cell adhesomes  whose acMoreover, SAM68 has been widely described as a regulator of alternative splicing. As all STAR family proteins, it contains a structural domain for binding of RNA composed of a KH RNA-binding module embedded within a conserved regulatory and signaling region (STAR domain). SAM68 has been shown to regulate the alternative splicing of CD44 in a signal-dependent manner  and the In light of the signal relay functions of SAM68 and its presence in integrin adhesion complexes, we set out to elucidate the role of SAM68 in endothelial cell-ECM interactions. We surveyed the spatio-temporal distribution of the RBP following integrin activation and analyzed its participation in integrin signaling and adhesion maturation. We show transient localization of SAM68 near nascent adhesion sites where it participates in cytoskeletal remodeling and local delivery of β-actin mRNA, which is known to contribute to adhesion site stabilization and growth. Moreover, we demonstrate that SAM68 orchestrates cell-matrix interactions by regulating the expression of key perivascular ECM components and assembly of the subendothelial matrix, a structure that provides important instructive signals for cell migration and morphogenesis. We propose that these coalescent activities of SAM68 play a key role in the adaptation of endothelial cells to their extracellular environment.To determine whether SAM68 is involved in the regulation of the adhesive phenotype of endothelial cells, we performed loss-of-function studies in primary cultured HUVECs by RNA interference. Transfection of two different siRNAs directed against human SAM68 transcripts efficiently decreased SAM68 protein levels (up to 90%), compared to those obsered in control siRNA-transfected cells . Followifocal complexes form at the cell periphery and rapidly disassemble or mature into more elongated focal adhesions, as force is applied upon linkage of adhesion components to the actin cytoskeleton. Long fibrillar adhesions that arise by translocation of α5β1 integrins out of focal adhesions along growing FN fibers on the cell surface are sites of FN fibrillogenesis in mesenchymal cells. Analyisis of the size distribution of vinculin-containing adhesions revealed a decrease in the proportion of long adhesions (>2 µm) in SAM68-depleted cells compared to control cells, and an increase in the proportion of the smallest adhesions  antisense to the Sam68-binding sequence of β-actin mRNA , previouIn endothelial cells, stabilized focal adhesions elongate and mature into fibrillar adhesions spanning the ventral cell surface. The accumulation of fibrillar adhesions can be visualized by immunostaining of integrin α5β1 . ConsistFibrillar adhesions are sites of FN fibrillogenesis. Thus, the sparsity of fibrillar adhesions in SAM68-depleted cells prompted us to investigate the ability of these cells to deposit a FN matrix. As illustrated by immunostaining of FN in endothelial cells plated on uncoated glass coverslips , depletiFN1 gene transcription. To test this, we generated a reporter construct (pFN) containing a 3.5 kb sequence encompassing the Src signaling-responsive FN1 gene promoter region  was normalized to the expression of total FN (tFN) transcripts . SelectiConsidering the major impact of SAM68 depletion on FN production and fibrillogenesis, and the well-documented role of FN on the assembly of higher order ECM networks, we next addressed the role of SAM68 in regulating the expression and organization of other components of the endothelial cell basement membrane. As shown in This role of SAM68 in subendothelial matrix synthesis and assembly suggested that SAM68 may participate in matrix-dependent modulation of endothelial cell adhesive behavior. To evaluate this, we compared the adhesion of control or SAM68-depleted cells to their own matrix by subjecting confluent monolayers to repeated rounds of mild trypsin digestion. siRNA-transfected cells were plated for 48 hr prior to protease treatment. Enumeration of detached cells in each fraction of diluted trypsin revealed that 80% of the SAM68-depleted cells detached from the matrix within 10 min of treatment whereas only 40% of control siRNA-transfected cells were detached at this time . Taken aProper organization and scaffolding of matrix components in the endothelial basement membrane are required for efficient capillary formation during angiogenesis and for maintenance of vascular homeostasis. Thus, we next assessed the functional consequences of SAM68 depletion on the motile behavior of endothelial cells in 2 and 3D contexts. In a 2D setting, migration of individual cells in sparse cultures was followed by time lapse video microscopy. As shown in We next interrogated the regulation of angiogenic behavior of endothelial cells by SAM68 in a 3D sprouting assay. To do so, Cytodex3 beads fully covered with siCTRL- or siSAM68-transfected cells were embedded in fibrin gels for 48 hr prior to confocal imaging of sprout formation. As shown in Here, we report that the RNA binding protein SAM68 regulates spreading, migration and the angiogenic phenotype of endothelial cells. First, we showed that SAM68 is localized near polymerizing actin in spreading cells where it contributes to the stabilization and growth of nascent integrin adhesions by local regulation of integrin signaling and delivery of β-actin mRNA transcripts. Further, at the cellular and multi-cellular levels we found that SAM68 contributes to the conditioning of the adhesive environment of cells by enhancing the expression and/or deposition of principal vascular basement membrane components which, in turn, affect cell motility and capillary-like formation. These findings demonstrate an important link between SAM68 and the angiogenic phenotype of cultured endothelial cells via coordinated functions of the RBP at submembraneous adhesion sites and in the nucleus, as schematized in +/+ or SAM68-/- mice, as opposed to using a loss-of-function approach on the same cell population as reported here. In subsequent work, SAM68 was detected at the spreading edge MRC5 fibroblasts in actin-sheathed bleb-like structures termed Spreading Initiation Centers (SIC)  during early adhesion on a FN-coated substrate . Howeverrs (SIC) . These srs (SIC) . Althougrs (SIC)  and in mrs (SIC) . Here, urs (SIC) , tensin3rs (SIC)  and Csk rs (SIC) . In the The group of Singer showed that localization of β-actin mRNA near cell attachment sites by the RBP ZBP1 contributes to the stability of focal adhesions , and theCell migration involves a complex interplay between integrin signaling and ECM composition. The involvement of SAM68 in cell migration was previously addressed in studies with varying outcomes, depending upon the cell type examined and experimental design. In epithelial cells and fibroblast-like synoviocytes, SAM68 silencing was reported to decrease migration and invasion  whereas FN1 gene by interacting directly with its promoter. Similarly, in a recent study, the RBP FUS was shown to induce expression of collagen IV by binding to its promoter , suggesting that SAM68 regulates isoform diversity through its transcriptional activity. Indeed, functional coupling between splicing and transcription of nclusion . The notnclusion .Beyond regulating synthesis and assembly of FN, which orchestrates the deposition of major basement membrane components (i.e. collagen IV and perlecan) that are required for stability and maturation of this specialized ECM , our datSprouting angiogenesis involves pro-invasive endothelial tip cells that extend numerous filopodial protrusions and cohesive stack cells that ensure connections to the primary vessel. Our fibrin gel sprouting assay revealed that SAM68-deficient endothelial cells produce shorter capillary-like structures, as compared to control cells, with tip cells that form stunted protrusive structures and frequently detach from the stalks. These results suggest that both adhesion-stabilizing and ECM gene-regulatory functions of SAM68 are required for capillary-like formation, however we cannot exclude that additional functions of the RBP may be involved in this 3D setting.Finally, beyond the functions described above, it is tempting to speculate that SAM68 plays a role in integrin-dependent phagocytic processes in endothelial cells, which are implicated in clearance of pathogens, apoptotic cells and ECM debris during tissue remodeling. Such processes are investigated using FN-coated beads and utilize the same molecular machinery as integrin-mediated firm adhesion, as reviewed in .In sum, our study illustrates a striking example of the multifaceted functions of RBPs in dynamic adhesion and actin-dependent membrane remodeling processes. With a focus on SAM68 in endothelial cells, we described the dynamic localization and functions of this adhesome-associated RBP at integrin adhesion sites and in the nucleus. Our findings should provide impetus for future studies to further decipher the mechanisms underlying RBP-dependent regulation of angiogenic processes.Key Resources Table (Appendix 1). All raw data associated with this manuscript are available in the source data files. All materials generated in this study are available upon request.Plasma FN was from Corning  Alexa Fluor 488- or 647-conjugated phalloidin and Hoechst 33342 were purchased from Invitrogen . Reagents for the 3D sprouting angiogenesis assay  were purchased from Sigma-Aldrich . Antibodies and oligonucleotides used in this study are indicated in the Primary human umbilical vein endothelial cells (HUVECs) were prepared from fresh human umbilical veins as previously described  and mainWhere indicated, culture plates or glass coverslips were coated with 10 µg/ml plasma FN in PBS for 1 hr at 37 °C. After coating, plates were washed in PBS and air-dried under the culture hood for 10 min.FN1 promoter activity reporter construct, 3500 nucleotides located upstream of the transcription initiation of FN mRNA were amplify from HUVEC genomic DNA using KAPA HiFi HotStart  and cloned upstream of the luciferase coding sequence of the pGL3-Basic plasmid . The SAM68 expression plasmid was generated by insertion of the complete SAM68 coding sequence, amplified from HUVEC cDNA and cloned in the pcDNA3.1 plasmid . The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites. The SAM68 P358A mutant was generated by site-directed mutagenesis and the SAM68 ΔKH mutant was created by removal of the sequences encoding residues 157–256 from the cDNA fragment. All constructs were checked by full-length sequencing .To generate the Primary endothelial cells were transfected in Opti-MEM  with either scrambled siRNA or 2 different siRNAs targeting human SAM68 mRNA sequence at a final concentration of 20 nM using Lipofectamine RNAiMAX . Sequence design of SAM68 siRNAs was performed using the Web-based DSIR tool . One dayhttps://www.addgene.org/protocols/lentivirus-production/) in the HEK293FT packaging cell line. Viral particles were harvested after 36 hr of production and used to infect 106 low passage endothelial cells the same day.Lentiviral particles were generated according to standard Addgene protocol . Proteins were separated on 7.5% SDS–PAGE gels and transferred to Amersham Hybond PVDF membranes  overnight at 4 °C (30 Volts) in Dunn buffer. Membranes were saturated in PBS 5% non-fat dry milk for 1 hr at RT and incubated in PBS 3% BSA with primary antibody (listed in Appendix 1—key resources table) overnight at 4 °C. Secondary antibodies conjugated to Horseradish Peroxidase were incubated in PBS 5% non-fat dry milk for 1 hr at RT. Immunostained bands were detected using Clarity Western ECL . Densitometric analysis of the band were performed using FIJI software .5 HEK293 cells were plated in 12-well plates, transfected using CaCl2 . Briefly, endothelial cells were grown to 80% confluency, incubated in 1% formaldehyde for 10 min at room temperature and the reaction was stopped with 0.25 M glycine. After scrapping, cells were resuspended and lysed in 50 mM HEPES, 140 mM NaCl, 1 mM EDTA and 0.5% Triton X-100 for 30 min on ice. Cell lysates were sonicated  and immunoprecipitated using Magnetic Dynabeads  preincubated with nonimmune IgG  or anti-SAM68 antibody . After reversion of the cross-link, immunoprecipitated complexes were treated with RNase A and proteinase K. DNA fragments were recovered by phenol-chloroform extraction followed by ethanol precipitation and assayed by qPCR to determine the fold enrichment of RNA extraction was performed using the RNA XS extraction kit  and cDNAs were generated from 1 µg of RNA using a High-Capacity cDNA Reverse Transcription kit . qPCR was performed on a StepOnePlus System (Applied Biosystems) using the PowerUp SYBR Green Master Mix  and relative expression levels were calculated using the ΔΔCt method, normalized to RPL27a mRNA levels unless specified.5 siRNA-transfected HUVECs were plated in 6-well plates and grown for 48 hr. Cells were gently detached by adding 1 ml of a 1:150 diluted trypsin solution  for exactly 2 min at 37 °C. Fractions of detached cells were collected and 1 ml of fresh diluted trypsin was added to the wells. Twelve cycles of trypsinization were performed (no washing between steps) until all cells were detached. Cells collected in each fraction were counted and expressed as a percentage of total collected cells.2.5x108 particles/ml of Polybead Microspheres  were coated overnight at 4 °C with plasma FN (10 µg/ml) in PBS. Microbeads were then deposited directly on endothelial cells (30 beads per cell) which had been plated overnight on uncoated glass coverslips and serum-starved for 1 hr prior to bead addition. Cells were fixed for analysis 20 min after bead addition.A total of 5.10For wound healing assays, confluent monolayers of siCTRL- or siSAM68-transfected HUVECs were manually scratched using a plastic tip, rinsed with warm PBS and maintained in complete medium supplemented with 2% FBS and 20 mM HEPES . Video microscopy was performed as previously described  using a Sprouting of siRNA transfected cells was evaluated according to the procedure described in Cells grown on 18- or 25 mm round glass slides  were fixed in 4% paraformaldehyde / 5% sucrose at 37 °C, washed in PBS and permeabilized in PBS 0.5% Triton. Blocking of unspecific interactions was performed in PBS containing 4% BSA  for 1 hr at RT followed by overnight 4 °C staining with primary antibodies diluted in blocking solution. Cells were then washed thoroughly with blocking solution and incubated with respective secondary antibodies diluted 1/4000 in blocking solution at RT for 1 hr. A final step of extensive washes in PBS is performed before air drying of the samples and mounting in Prolong Gold antifade (Invitrogen P36930). Cells imaged using TIRF confocal microscopy were not mounted and left at °4 C in PBS.https://bitbucket.org/muellerflorian/fish_quant  were hybridized to endogenous β-actin mRNA according to the smiFISH protocol , using SUnless otherwise specified, confocal imaging was performed on a Nikon ECLIPSE Ti 100 x (SPINNING DISC) using Metamorph Acquisition software  using 40 X water 1.2 NA, or 100 X oil objective (NA 1.4) with the LASERs 405, 488, 561, 633 nm. For live cell imaging of eGFP-SAM68 /LifeAct Ruby sequence of 30–60 s duration were acquired on TIRF illumination mode every 100ms. Whole coverslip imaging of the ECM, was carried out using the PhenoImager HT (formerly Vectra Polaris) slide scanner in the 40 X mode .Statistical analyses were performed with either R  or PrismThe paired Student's t-test was applied, unless specified in the legend. Differences were considered to be statistically significant at p<0.05 (*), p<0.01 (**), p<0.001 (***) and data are expressed as mean ± SD. N=number of biologically distinct experiments; n=number of observations per condition.See Analysis of ECM deposition was performed on whole-coverslip scanned images with Halo bioimage analysis software . Briefly, the area corresponding to the fluorescent signal of each matrix component was segmented using the ‘Area Quantification FL’ plugin of the software and subsequent measurements (in µm²) were normalized to the number of cells on each slide. Number of cells was assessed by nuclei count after segmentation using the nuclei segmentation plugin of the software. This paper provides important evidence that the RNA binding protein SAM68 regulates endothelial cell migration through multiple mechanisms including localizing actin mRNA to focal adhesions and stimulating transcription of the fibronectin gene. The evidence is generally convincing, although the relative roles of transcription and RNA localization in SAM68 functions and the dynamics of RNA movement to adhesion sites remain unknown. The paper will be of interest to cell biologists investigating post-transcriptional regulatory mechanisms. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  General Statements [optional]The goal of our study was to evaluate the role of the RNA binding protein SAM68 in the regulation of cell adhesion and adaptation of endothelial cells to their extracellular environment. We showed that SAM68 depletion affected endothelial cell behavior by impairing adhesion site maturation and compromising basement membrane assembly.We are pleased that the reviewers found our study to be interesting, well written and clear, with findings that are supported by carefully designed experiments. Importantly, we would like to thank the reviewers for their careful analysis of our work and for their clear and constructive comments.FN1 gene transcription by SAM68 in endothelial cells involves direct interactions with the mRNA and promoter, respectively. This important point will be addressed with additional experiments in order to strengthen our hypothesis.One common query was whether the regulation of β-actin mRNA localization at adhesion sites, and FN1 gene transcription. Our response to this issue is discussed below and has been clarified in the revised manuscript.A second point that emerged from the reviews relates to the interdependence of SAM68 multi-layered effects on cell adhesions and Lastly, since in vivo studies are not feasible locally or in a reasonable timeframe, our claim that SAM68 tunes an endothelial morphogenetic program has been toned down in the revised manuscript. Nonetheless, our data clearly show that SAM68 is a major regulator of endothelial adhesion and conditioning of the subendothelial basement membrane.a significant advance towards understanding the multiple roles of RNA-binding proteins and their coordination in a study system with physiologically relevant connections”, as stated by Reviewer#3.Altogether, the proposed experiments and revisions will solidify our data and improve our study thus providing “Description of the planned revisionsTo answer critical points raised by the 3 referees, we plan to implement our work with 3 main sets of experiments:Set 1 of experiments: Analysis of direct interaction between SAM68 and b-actin mRNA by RIP in endothelial cells according to an improved version of published protocols and results from .Set 2 of experiments: Analysis of a direct interaction between SAM68 and the FN1 promoter by ChIP in endothelial cells according to published protocols and results from .Set 3 of experiments: Assessment of the dual functions of SAM68 and their interconnections by (i) FN rescue (expression of exogenous FN in SAM68-depleted cells) or (ii) by expression of SAM68 mutants.In addition, we are generating tools to address the dynamic localization of b-actin in endothelial cells following SAM68 perturbations in endothelial cells .Below, we describe how these sets of experiment will address Reviewers’ comments and queries in a point-by-point reply.Our point-by-point responses regarding the revisions, including the outcomes of our proposed sets of experiments, our new results and how we have incorporated them in the manuscript and figures of the final revised version are indicated below in bold blue text.Reviewer #1– The authors claim that SAM68 interacts with B-actin mRNA to delivery to sites of adhesion only based on siRNA-mediated knockdown experiments. Is the binding of SAM68 to B-actin dynamic process that changes with time? The authors could perform RIP experiments at different stages of cell adhesion – from early points when SAM68 is peripheric to later stages when it homogeneously distributed – to show a potential dynamic interaction with B-actin mRNA.β-actin mRNA has been previously identified as a direct target of SAM68 in several published works performed by different groups . SAM68 binding site has been mapped to a 50 nt length sequence located in the 3’UTR of β-actin mRNA but direct binding of SAM68 onto β-actin mRNA has never been shown in endothelial cells. To this end, we will perform RIP experiments (Set 1 of experiments) to first identify direct recruitment of SAM68 to β-actin mRNA in endothelial cells (as suggested by reviewer 2 as well). Secondly, to address the dynamics of SAM68 interactions with β-actin mRNA we will assess direct interactions of SAM68 with β-actin mRNA at different stages of cell adhesion. These experiments will be conducted using an adapted version of the published SAM68 RIP protocol .In order to demonstrate the direct involvement of SAM68 in β-actin mRNA localization at ectopic adhesion sites in endothelial cells, we transfected cells with antisense blocking oligonucleotides that target the SAM68-binding site on b-actin mRNA and are known to abrogate SAM68 and β-actin mRNA interaction. As shown in new Figure 4C and discussed in the text of the revised manuscript, these blocking oligonucleotides significantly decreased the localization of β-actin mRNA in adhesion sites thus strengthening our claim of SAM68-mediatd delivery of mRNA to nascent adhesion sites and indicating a direct effect of SAM68 via interaction with b-actin mRNA.Regarding the dynamics of SAM68 interaction with b-actin mRNA, RIP experiments performed at different stages of cell adhesion did not reveal any significant differences and therefore we have not included these results in our revised manuscript.– The article would substantially benefit from live visualisation of B-actin localisation with MS2 tagged transcripts in SAM68 knockdown contexts. This would solidify the proposed mRNA delivery SAM68-mediated mechanism. Although this should not be hard to carry out given the availability of MS2-labelled animals, I understand access to the tools may constitute a major hurdle.in cellulo experiments in endothelial cells to visualize MS2-β-actin reporters , in presence of control or SAM68 binding site-directed antisense blocking oligonucleotides, as previously described .As mentioned by Reviewer 1, access to MS2-labelled animals and carrying out in vivo experiments in mouse endothelial cells would be a roadblock for our team in the context of this work. Nonetheless, we fully agree that live visualization of β-actin mRNA recruitment at adhesions would solidify our hypothesis. Therefore, we are currently setting up Despite our attempts, it was not possible to generate stable MS2- β-actin reporter expressing primary endothelial cells to visualize live SAM68-dependent β-actin localization. However, the transfection of SAM68 binding site-directed antisense blocking oligonucleotides  has allowed us to confirm the direct implication of SAM68 in endogenous β-actin mRNA delivery to adhesion sites in endothelial cells.Minor comment:– Could the authors run the eGFP-SAM68 movies for longer periods to show the dynamic localisation of the protein during spreading? These experiments would support the data based on fixed material.We thank Reviewer 1 for this suggestion and will adjust our imaging pipeline for longer time acquisitions taking caution not to impact cell dynamics due to extended laser exposure.with confidence the dynamic localization of the protein during spreading.We have performed live imaging of spreading eGFP-SAM68/LifeAct-Ruby expressing cells at longer times in an attempt to capture the dynamics of the rapidly-moving SAM68 particles. Unfortunately, and despite our attempts to adjust our imaging pipeline, the illumination of these cells for more than a few minutes induces phototoxicity (retraction of cell edges). Therefore, we are unable to include additional movies for longer periods to capture Reviewer #2– The authors reference previous work defining SAM68 as a β-actin mRNA interacting protein, however, experiments confirming this in endothelial cells and that this occurs during normal focal adhesion assembly are important.This concern will be addressed by Set 1 of experiments (RIP assays) as described in our response to the comments of Reviewer 1.– Likewise, experiments addressing how important this action is for focal adhesion function are critical. For example, the β-actin RNA-binding site of SAM68 could be identified and perturbed to assess the direct impact of this mRNA delivery on FAK-Y397 phosphorylation, focal adhesion assembly, adhesion, cell spreading and migration/sprouting. Without these or similar experiments, the importance of SAM68-mediated β-actin mRNA delivery is unknown.The β-actin RNA-binding site of SAM68 has previously been identified  and antisense blocking oligonucleotides designed to target this sequence have been shown to abrogate SAM68 recruitment onto β-actin mRNA in neurons . In order to determine whether SAM68 delivery of β-actin mRNA is directly involved in focal adhesion assembly and signalling, we will use the published antisense oligonucleotides to block SAM68 recruitment and assess FAK-Y397 phosphorylation in our bead model.– Indeed, if this is not important for FAK-Y397 phosphorylation and focal adhesion assembly, then experiments need to be designed to assess how SAM68 achieves FAK phosphorylation/maturation to provide any significant insight into SAM68 function.To address this point, we have generated an RNA binding mutant of SAM68 (KH domain) for analysis of FAK phosphorylation using the bead assay. Importantly, SAM68 is a multi-domain protein that harbors protein/protein interaction domains (SH2 and SH3 binding domains) and it is known to act as a scaffolding protein, in TNFRα signaling for instance . Therefore, we have also generated lentiviral constructs containing mutations in the SH2 or SH3 binding domains of SAM68 to interrogate its potential signaling adaptor function.Our responses to the above comments of this Reviewer are indicated below.Regarding the interaction of SAM68 and b-actin mRNA in endothelial cells:As indicated in our response to Reviewer 1, we believe that our new results showing that the expression in endothelial cells of blocking antisense oligonucleotides targeting the SAM68 binding site in the 3′ UTR of β-actin mRNA decreases β-actin mRNA delivery to adhesion sites confirm that SAM68 is a b-actin mRNA interacting protein in these cells. Further, they indicate that this interaction is directly involved in β-actin mRNA localization during focal adhesion assembly .Regarding the importance of this action (SAM68 mRNA binding) for focal adhesion function:To interrogate how important SAM68 mRNA-binding activity is for focal adhesion signaling we generated functional mutants of the protein, depicted in new Figure 3D. Using an RNA binding-deficient mutant of SAM68 (SAM68 DKH) we first determined that RNA binding activity of SAM68 is critical for integrin signaling, assessed by FAK-Y397 phosphorylation. Further we determined that FAK-Y397 phosphorylation also involves the scaffolding activity of SAM68, using a SH3-binding mutant of SAM68 (SAM68 P358A) with impaired Src binding. These results have been included in the revised Figure 3D and detailed in the text.– The data as presented suggest that a key function of SAM68 is to drive fibronectin (and perhaps other ECM gene) transcription. However, more experiments are needed to validate this conclusion. For example, increased FN1 promoter activity in luciferase assays may be an indirect consequence of feedback to the promoter upon SAM68-mediated action on, amongst other possible actions, focal adhesion signaling, FN transcript splicing or ECM remodeling. Experiments confirming that SAM68 interacts with the endogenous ECM gene promoter would be critical (e.g. via ChIP), as would disruption of the trans-activating action of SAM68 to directly assess the impact of this function  on focal adhesion assembly, adhesion, cell spreading and migration/sprouting.FN1 gene in these cells. Therefore, we will perform these experiments (Set 2) according to a published SAM68 ChIP protocol  which allowed for the demonstration of specific recruitment of SAM68 onto P21 or PUMA promoters, as well as its transcriptional co-activating activity .We fully agree that ChIP experiments to identify recruitment of SAM68 onto the endogenous FN1 promoter in endothelial cells would be required to confirm direct transcriptional activation of the FN1 promoter activity in the luciferase assay shown in Figure 1F on HEK293 cells, we would like to point out that, in addition to their high transfection efficiency, HEK293 cells were chosen for this assay because they display nearly undetectable expression of FN and they are unable to assemble the molecule . Thus, our results using this system support a direct effect of SAM68 on FN promoter activity. This information has been added to the revised text.Regarding possible indirect effects of FN1 promotor have been added to the revised version of the manuscript .Results of ChIP experiments showing direct recruitment of SAM68 to the – In parallel, rescue experiments to determine how recovery of endothelial FN expression impacts adhesion, cell spreading, and migration/sprouting (upon SAM68 knockdown) would determine how important this action is to control of endothelial cell behavior.Our previous published data showed that autocrine FN expression regulates adhesion, spreading and migration of endothelial cells and that differences in FN expression levels affect assembly of the protein . In SAM68-depleted cells, with compromised FN expression, the rescue of FN expression should allow us to uncouple SAM68 functions at adhesion sites from its role as a transcriptional regulator of FN expression (Set 3 of experiments). Expression of exogeneous FN in SAM68-depleted endothelial cells will be performed using lentiviral FN expression constructs described by our team .– Likewise, experiments designed to determine if broader disruption of COL8A1, POSTN, FBLM1 and BGN expression are direct  would be important to understand SAM68 function.COL8A1, POSTN, FBLN1 and BGN mRNAs upon the rescue of FN expression in SAM68-depleted cells.The same set of experiments (Set 3) will be used to analyze by qRT-PCR the expression of We were unable to stably express exogenous FN using the above-mentioned expression constructs in our primary endothelial cell cultures and thus unable to explore possible indirect effects (due to FN disruption) of SAM68 knockdown on expression of other basement components.– Loss of SAM68 expression in other cell types is known to perturb migration, whereas migration is enhanced in endothelial cells upon SAM68 knockdown. Why would this be the case? Is it that the proposed negative impact of FN production on motility is greater than the positive impact of SAM68 focal adhesion dynamics in endothelial cells versus other cell types? Exploration of the relative impact of these proposed dual functions  is critical to make sense of these somewhat conflicting observations.negative impact of SAM68-stimulated FN production on motility and the positive impact of SAM68 on focal adhesion dynamics in endothelial cells, is very interesting. Set 3 of experiments, which includes expression of exogenous FN and assessment of cell motility in SAM68-depleted endothelial cells, should allow us to clarify this issue.This point relating to the balance between the versus no coat) could account, at least in part, for the differences observed in other studies, compared to the present work and previous reports on endothelial and glioblastoma cells .As mentioned in our response to the previous point, we were unable to stably express exogenous FN using the above-mentioned expression constructs in our primary endothelial cell cultures to assess the relative impact of the dual functions of SAM68 on cell migration. However, as indicated in the Discussion section (p. 14), we maintain that experimental conditions . Additional work exploring the impact of SAM68 phosphorylation on focal adhesion dynamics and ECM gene expression/remodeling (e.g. using phospho-mutants) in this manuscript would have strengthened the message.The regulation of SAM68 activity by phosphorylation is a complex question as SAM68 has multiple sites of phosphorylation by serine/threonine and tyrosine kinases. One of these sites (Y440) is a known substrate of Src, a major kinase activated at the cell membrane during adhesion. We are currently generating a Src phosphorylation mutant of SAM68 (Y440F) which could be used to address the impact of SAM68 phosphorylation on integrin signaling and ECM gene expression/remodeling.As indicated in our previous response to this comment, regulation of SAM68 by tyrosine and serine/threonine phosphorylation  is complex and difficult to dissect with only one mutant, and elucidating this complex regulation is beyond the scope of the present study.However, in light of the implication of SAM68 in FAK autophosphorylation on its Src binding site (tyrosine 397), and the importance of FAK-Src signaling in integrin signaling we generated a Src-binding-deficient mutant of SAM68. Interestingly, expression of this mutant in endothelial cells slightly increased the number of pFAK-Y397 foci around beads, as shown in new Figure 3D. These results are in line with previous studies reporting a role for interactions between SAM68 and Csk  in the downregulation c-Src following cell adhesion to FN. Further, they confirm a role, albeit complex, for SAM68 in the regulation of integrin signaling and focal adhesion dynamics. (See text p. 8 and 13 and for detailed discussion.)Reviewer #3– The authors describe the observed phenotypes as resulting from 'coalescent activities' of SAM68 that play a role in the adaptation of ECs to the extracellular environment. However, it is unclear whether and which of the observed effects result from direct local functions of different SAM68 pools, versus reflecting indirect downstream consequences of one major function. For example, the effects on transcription could be a result of altered adhesion signaling and might occur independently of nuclear SAM68. Or the effects on adhesions could be an indirect consequence of altered transcription of ECM genes, independent of the transient accumulation of SAM68 at the periphery. To support that these are distinct and direct SAM68 functions, the authors would have to provide more evidence for the involvement of SAM68 in the studied processes (e.g. is SAM68 observed by CHIP at promoter regions of ECM genes whose transcription is affected?)FN1 promoter (Set 2 of experiments).As recommended by Reviewer 2 as well, we will perform ChIP experiments to document the direct recruitment of SAM68 onto the As indicated in the response to Reviewer 2, the ChIP experiments have been performed and included in Figure 5G– Try to uncouple them to assess their relative contributions and potential connections in the observed phenotypes e.g. it would be informative to attempt to rescue the knockdown phenotypes with mutants of SAM68 that cannot be imported into the nucleus or that cannot bind RNA or that cannot be phosphorylated by SrcSet 3 of experiments should allow us to uncouple dual functions of SAM68 in endothelial cells. In these experiments, integrin signaling defects will be evaluated in SAM68–depleted cells following the rescue of FN expression. Persistence of the adhesion site defect would indicate that transcriptional activity and adhesion site regulation by SAM68 are distinct events. Moreover, as indicated above, we are generating lentiviral constructs of SAM68 mutants with impaired ability to bind RNA or be phosphorylated by Src (Y440F), in order to assess their effect on integrin signaling.As indicated in the response to Reviewer 2, we were unable to successfully carry out FN rescue experiments proposed in set 3 of experiments. However, as mentioned in our response to Reviewer 2, we were able to examine the functional consequences of rescuing SAM68 knockdown with mutants deficient in RNA binding/processing or impaired for Src kinase binding/signaling. We believe that the results of these experiments , together with results of the ChIP experiments  described in the revised manuscript support distinct and direct functions of SAM68.Minor comment:Also, is the effect of SAM68 depletion on pY397-FAK levels local and/or transient? it would be useful to present data on the total amount of pY397-FAK (by IF or western) in control and si-SAM68 cells at early and late stages of spreadingThis point is very interesting and will be tested at early vs late stage of spreading.Due to time constraints and prioritization, we were not able to undertake these longitudinal analyses.Description of the revisions that have already been incorporated in the transferred manuscriptReviewer#1– The authors state that "…both submembranous functions […] and nuclear functions […] of SAM68 contribute to the morphogenetic phenotype of angiogenic endothelial cells. Some caution must be taken, as all previous data were obtained from 2D experiments. At this stage it cannot be excluded other mechanisms involved in 3D migration.We fully agree with this reviewer’s comment and we have modified the manuscript to take into account the fact that we cannot exclude other mechanisms of action for SAM68 in 3D endothelial cell sprouting experiments However, it is noteworthy that the migration per se of individual cells is not measured in our 3D experiments.Minor comments:– In Figure 1F, there is a drop-in luciferase activity in cells transfected with higher amounts of vector, rather than an increase with SAM68. Why?The luciferase reporter assay is a convenient and well-accepted means of evaluating promoter activities, however, it requires the transfection of increasing amounts of expression plasmids, which often contain strong promoters such as CMV (in our case). Depending on the experimental conditions, a drop in luciferase activity is often observed, due to titration of general transcriptional factors. In our experiments shown in Figure 1F, despite the observed drop in luciferase activity in pcDNA 3.1-transfected cells, transfection of increasing amounts of the SAM68 expression vector induced a significant increase in luciferase activity.– The authors claim (and rightly so) that plasmids are hard to transfect into HUVECs when describing luciferase reporter assays. However, they express eGFP-SAM68 (presumably from a plasmid).eGFP-SAM68 was delivered and expressed in endothelial cells using a lentiviral vector (this has been specified in the revised manuscript: legend to Movie supplement 1). Although eGFP-SAM68 is successfully expressed, the efficiency of infection is a bit low. Thus, this method is adequate when experiments require observations at the single cell level, such as imaging of endothelial cells expressing eGFP-Sam68. However, the low infection efficiency makes it unsuited for the observation of global effects on the cell population, as is the case for a luciferase assay in which all cells from a given experimental condition are lysed.– Some experimental details in the figure legends could be restricted / moved to the methods section.– Some typos and British/American spelling inconsistencies  need to be corrected throughout the manuscript.– Statistical analysis details could be mentioned in figure legends.– In page 11, "… proposed to be involved in regulation of early cell adhesion processes and spreading" needs referencing.– Y axes in some graphs do not start at 0, which may mislead visual interpretations.– "Figure 2—figure supplement 1" in page should read "movie supplement 1"We thank Reviewer #1 for these comments which have all been taken into account. Appropriate changes/corrections have been made in the revised version of the manuscript.Reviewer#2– The title of the manuscript states that SAM68 modulates the morphogenetic program of endothelial cells, yet there are no studies of blood vessel morphogenesis described by this work. Ultimately, in vivo studies of vessel development in SAM68 mutant mice would be required to be able to make this claim.We agree that only endothelial cell morphogenesis, and not blood vessel morphogenesis, has been addressed in this study. In light of the reviewer’s recommendation to tone down claims that SAM68 tunes an endothelial morphogenetic program, we have modified the revised manuscript text and title.– Place the work in the context of the existing literature .SAM68 has previously been identified as an RNA-binding protein associated with the 'adhesome' that regulates cell motility . Here, Rekad and colleagues also probe the action of SAM68 in endothelial cell migration, but find this to be enhanced upon SAM68 knockdown – unlike previous studies demonstrating a reduction in motility in similar experiments in other cell types. Indeed, a detailed discussion of this discrepancy would have been appreciated.As recommended by Reviewer #2, we have included a more detailed discussion of this point in the revised manuscript.Reviewer #3Figure 3C: Is the n=3 indicative that only 3 beads were analyzed? Given the relatively small difference, a larger sample size would be useful.We thank the Reviewer for pointing out this mistake. Three independent experiments have been performed with quantification of at least 12 beads for each condition. The manuscript has been corrected accordingly (N=3).Page 5-6: The statement 'nearly all adhesion sites in SAM68-depleted cells remained smaller than 0.75 um' doesn't seem to accurately reflect the data presented in the right panel of Figure 1C.2) of average adhesion size. Nearly all adhesion sites in SAM68-depleted cells remained smaller than 0.75 µm2We have modified the units 00456-8) had shown the presence of SAM68 in SICs. Why do the authors believe that the presence of SAM68 in the periphery in endothelial cells does not mark the formation of SICs in these cells?Spreading Initiation Centers (SICs) are described as structures involved in the early step of adhesion which contain SAM68, along with other RNA binding proteins  in MRC5 cells. In the same paper, to test whether SICs are a general feature of cell adhesion, authors evaluated the presence of SICS during adhesion of several other cell including endothelial cells (HUVEC). Among the 6 types of cells tested, SICs were not observed in nonfibroblastic cell types. In accordance with this study, we did not observe SICs, as defined by deHoog et al., in endothelial cells plated onto FN– In Shestakova et al. 2001 (doi.org/10.1073/pnas.121146098), decreased localisation of B-actin mRNA leads to reduced persistence of direction of movement. Was this measured? Is this not seen here because SAM68 is only responsible for B-actin mRNA localisation at early stages of adhesion?We thank Reviewer 1 for this comment. After re-analysis of our migration data we did not detect a significant effect on the persistence of migration in our experimental conditions. This could indeed reflect the temporal regulation by SAM68 of b-actin mRNA localization at the leading edge of cells, although we cannot exclude additional defects caused by SAM68 depletion on adhesion stability and lammelipodial protrusion and consequently cell polarity and directional motility.– Although the authors claim that altered ECM deposition in SAM68 deficient cells results from altered transcription, they do not address potential misregulation of translation and secretion.We did not address misregulated translation here as FN mRNA levels were significantly decreased in SAM68-depleted cells. The decreased transcript levels were accompanied by decreased protein levels. Upon depletion of SAM68, we detected less FN in both “soluble” (conditioned medium) and “insoluble” (ECM-associated) forms, as shown in the western blots of Figure 4—figure supplement 1. We do not believe that SAM68 silencing impacts FN secretion, as we did not observe differential retention of FN in the cytoplasm of SAM68-depleted cells compared to control cells by immunostaining . Rather, FN staining was strictly fibrillar (ECM-associated) in both control and SAM68-depleted cells, and the intensity profile baseline values were similarly low. This point has been added to the revised manuscript.– In fact the highlight that whilst the level of some mRNAs encoding basement membrane proteins do not decrease in the absence of SAM68, their incorporation was severely affected. This is worth exploring to strengthen the manuscript.This issue was not addressed for other basement membrane components. However, the dichotomy in expression and matrix incorporation of certain basement membrane components is most likely due to the sequential and hierarchical nature of ECM assembly. FN is one of the earliest ECM proteins to be assembled and observations from multiple laboratories have shown that FN orchestrates the assembly of multiple matrix components ), including COLIV ).– Whilst the data presented in figure 7 is convincing, some more detailed mechanistic analyses could help further comprehend 2D and 3D behaviours. Could it be that the nuclear and cellular roles of SAM68 are somewhat decoupled depending on the environment? Could the RNA localisation functions have a critical role in endothelial sprouting and not so much in 2D migration? Some insights are needed to address these questions and wrap up some loose ends. In its current form, this section of the manuscript is too vague.It is known that 2D and 3D culture conditions induce differences in cell behavior, notably through differences in the physical (rigid vs pliable) and biochemical (plastic vs fibrin gel) nature of the environments that differentially regulate mechanotransduction, integrin signaling, cell polarity, etc. Here, we show that migration of endothelial cells on rigid 2D substrates is increased upon SAM68 depletion. On the other hand, the ability of cells to align in capillary-like cords and invade a 3D environment is reduced. Mechanistically, effects of SAM68 on FN production and ECM assembly are likely involved in both contexts by providing an adhesive substrate that restricts cell motility in 2D, or bridges neighboring cells and promotes cell survival in 3D. The purpose of performing the sprouting assay presented here in addition to cell migration assays was not to compare the same functions of SAM68 in these 2 different contexts but rather to illustrate that SAM68 controls endothelial cell behavior in both 2D and 3D environments and thus could significantly impact angiogenesis.Reviewer#2– Many of the findings are rather superficial or observational, and a detailed mechanistic understanding of SAM68 function is lacking. For example, loss of SAM68 expression reduces β-actin mRNA recruitment to sites of fibronectin-coated bead adhesion, but how is this regulated and what is its impact on focal adhesion dynamics?Both the role of β-actin mRNA localization on cell adhesion dynamics and the impact of reducing this localization have been extensively documented , or  and references therein. In particular, a specific RNA binding protein called ZBP1 has been shown to localize actin mRNA near focal adhesions  by a Src kinase-dependent mechanism .ReferencesJ Cell Sci 123:3989–3999. doi:10.1242/jcs.073346Cseh B, Fernandez-Sauze S, Grall D, Schaub S, Doma E, Van Obberghen-Schilling E. 2010. Autocrine fibronectin directs matrix assembly and crosstalk between cell-matrix and cell-cell adhesion in vascular endothelial cells. Dallas SL, Chen Q, Sivakumar P. 2006. Dynamics of Assembly and Reorganization of Extracellular Matrix ProteinsCurrent Topics in Developmental Biology. Academic Press. pp. 1–24. doi:10.1016/S0070-2153(06)75001-3Cell 117:649–662. doi:10.1016/s0092-8674(04)00456-8de Hoog CL, Foster LJ, Mann M. 2004. RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers. 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Spatial regulation of β-actin translation by Src-dependent phosphorylation of ZBP1. Nucleic Acids Res 30:5452–5464. doi:10.1093/nar/gkf673Itoh M, Haga I, Li Q-H, Fujisawa J. 2002. Identification of cellular mRNA targets for RNA-binding protein Sam68. Genes Dev 26:1885–1890. doi:10.1101/gad.190413.112Katz ZB, Wells AL, Park HY, Wu B, Shenoy SM, Singer RH. 2012. β-Actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration. J Cell Biol 127:441–451. doi:10.1083/jcb.127.2.441Kislauskis EH, Zhu X, Singer RH. 1994. Sequences responsible for intracellular localization of β-actin messenger RNA also affect cell phenotype. Proc Natl Acad Sci U S A 110:3125–3130. doi:10.1073/pnas.1209811110Klein ME, Younts TJ, Castillo PE, Jordan BA. 2013. RNA-binding protein Sam68 controls synapse number and local β-actin mRNA metabolism in dendrites. Nucleic Acids Res 44:8726–8741. doi:10.1093/nar/gkw582Li N, Richard S. 2016. 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Food sensitization in early life identifies children at risk of developing allergic diseases. We investigated the sensitization to cow milk (CM), egg whites, and wheat. Newborns and infants under 3 years of age with available specific immunoglobulin E (sIgE) data were identified. A retrospective survey was conducted using data from the Chang Gung Research Database. Perinatal characteristics, such as singleton or multiples in a single pregnancy, parity, meconium staining, maternal age, spontaneous delivery or cesarean section, meconium passage, weeks of gestation, birth length, body weight, head and chest circumferences, and season, were obtained. The data on sIgE were collected, and a logistic regression model was used to determine the odds of sensitization. Positive sIgE for CM and egg whites was more likely to occur in boys than in girls. Early-life egg white and wheat sensitization was associated with increased birth body length and weight. A multivariate analysis indicated an association between egg white sIgE positivity and logarithmic total IgE. Higher total IgE levels and younger age were associated with egg white sensitization, and elevated weight and length at birth were linked to food sensitization, particularly to egg whites and wheat. Early-life food sensitization has identified children who are at high risk for subsequent allergic diseases . In partFood allergies are complex diseases characterized by genetic and environmental factors ,5. Cow mThis retrospective study used data from the Chang Gung Research Database to investigate the association between perinatal characteristics and sIgE against CM, egg whites, and wheat from 2015 to 2019. The research protocol was approved by the Ethics Committee of the Chang Gung Memorial Hospital (202001016B0). As shown in Specific IgE between 0 and 0.34 kUA/L using ImmunoCAP or 0 and 26 LUs using MAST was considered negative ,19. For t-test was applied to explore the effect of gestational age, birth weight/length, and maternal ages divided into two groups (< 40 years and ≧ 40 years). One-way analysis of variance was used to compare the three groups of maternal age . Odds ratios (ORs) were adjusted for confounding variables and calculated using negative sensitization as the reference category. Adjustments with multivariable logistic regression were allowed for possible relevant confounders and factors showing a potential influence in univariate analysis, such as age, sex, logarithmic (log) total IgE, and birth body length, to identify the independent factors for allergen sensitization in infants. Analyses were performed using the Statistical Analysis System Package .Categorical results were presented as numbers and continuous results as means ± standard deviation (SD) or median (range). We used chi-square or the Fisher’s exact test to detect significant differences between sensitization to CM, egg whites, or wheat, and characteristics, such as sex, mode of delivery, parity, birth season, maternal age, and other perinatal factors. Student p = 0.012; p = 0.286). The children with positive sIgE to CM had higher total serum IgE levels than the children with negative sIgE (median 178 U/mL (range 11.9–3749.0) in the group with positive sIgE; 59.2 U/mL (range 2.1–4349.0) in the group with negative sIgE; p < 0.001 of both log and arithmetic calculations). We found significantly higher log total IgE levels in boys when we divided our data into groups with negative and positive CM-sIgE  [p = 0.613). We hypothesized that there was an association between perinatal characteristics and the odds of sensitization to CM and found that CM-positive sIgE was not associated with cesarean delivery, number of fetuses, parity, meconium staining, maternal age, birth length and weight, head and chest circumferences, meconium passage, or birth season (p > 0.05). Similar results were observed when the patients were grouped according to sex. Independent factors for sIgE positive to CM were elevated total IgE or log total IgE values (adjusted odds ratio (aOR) 1.001, p < 0.001; aOR 3.820, p < 0.001) and not age, birth body length, or sex  tested positive for the sIgEs against CM. In total, 32.1% and 8.2% of the children tested positive for egg whites- and wheat-sIgE, respectively . A total= 0.046) . The mea, or sex .p = 0.001, p = 0.207). The mean levels (SD) of gestational age in children with negative and positive egg white-sIgE were 38.5 ± 1.6 weeks and 38.5 ± 1.3 weeks (p = 0.629), respectively. Two hundred and seventy-six (25.1%) children were born by a cesarean section (p = 0.796 between the groups with negative and positive egg white-sIgE). Furthermore, greater birth body lengths and weights were associated with significantly higher odds of egg white and wheat IgE positivity  but not sex . Our findings indicate that a greater proportion of boys (36.3%) than girls (26.4%) aged 0–3 years had egg white sensitization (p > 0.05). Maternal age was similar in the negative and positive groups whether we divided it into two groups . Singleton or multiple pregnancies, parity, meconium staining, maternal age, normal spontaneous delivery or cesarean section, birth head and chest circumferences, meconium passage, birth season, and weeks of gestation were not associated with egg white sensitization. A univariate analysis showed that the developments of sIgE to CM, egg whites, or wheat were not related to birth season (p = 0.006 after the Bonferroni correction for multiple comparisons) but not wheat . Children with wheat sensitization had similar odds of developing the above-mentioned allergic diseases compared to those without wheat sensitization (p > 0.05)  .p = 0.003; aOR 1.001, 95% CI 1.000–1.001, p < 0.0001; p = 0.012; aOR of log total IgE, 1.002; 95% CI 1.002–1.003, p < 0.0001; p < 0.0001); however, this was not significant before 2015 . Despite the large number of participants in the database study, the present study had some limitations. The first limitation was rooted in false negatives, which were non-IgE-mediated food allergies ,50. The However, the influence of birth factors on food sIgE levels remains controversial. In summary, higher total IgE levels and a younger age were associated with egg white sensitization. We found that increased birth weight and length were associated with food sensitization, particularly to egg whites and wheat.


Rapidly and efficiently identifying critically ill infants for whole genome sequencing (WGS) is a costly and challenging task currently performed by scarce, highly trained experts and is a major bottleneck for application of WGS in the NICU. There is a dire need for automated means to prioritize patients for WGS.Mendelian Phenotype Search Engine (MPSE).Institutional databases of electronic health records (EHRs) are logical starting points for identifying patients with undiagnosed Mendelian diseases. We have developed automated means to prioritize patients for rapid and whole genome sequencing (rWGS and WGS) directly from clinical notes. Our approach combines a clinical natural language processing (CNLP) workflow with a machine learning-based prioritization tool named MPSE accurately and robustly identified NICU patients selected for WGS by clinical experts from Rady Children’s Hospital in San Diego (AUC 0.86) and the University of Utah (AUC 0.85). In addition to effectively identifying patients for WGS, MPSE scores also strongly prioritize diagnostic cases over non-diagnostic cases, with projected diagnostic yields exceeding 50% throughout the first and second quartiles of score-ranked patients.Our results indicate that an automated pipeline for selecting acutely ill infants in neonatal intensive care units (NICU) for WGS can meet or exceed diagnostic yields obtained through current selection procedures, which require time-consuming manual review of clinical notes and histories by specialized personnel.The online version contains supplementary material available at 10.1186/s13073-023-01166-7. It is estimated that 7 million infants are born worldwide with genetic disorders each year . AdmissiCurrent estimates suggest that around 18% of neonates admitted to the NICU harbor a Mendelian disease, and rWGS diagnostic rates in this population are over 35% , 5. RapiPhenotype descriptions are crucial components of the WGS diagnostic process, and many tools exist for combining phenotypic terms with WGS data to prioritize disease-causing variants –10. CurrCare providers emphasize the importance of clinical notes for informing disease diagnosis, and HPO-based phenotype descriptions are generally compiled through manual review of these free text documents. Unfortunately, this is a time-consuming process that requires highly trained experts and is a major bottleneck for application of WGS in the NICU , 13.Natural language processing (NLP) is a class of computational methods for generating structured data from unstructured free text. Recent work has begun to explore the utility of using clinical natural language processing technologies (CNLP) to automatically generate descriptions directly from clinical notes, with several groups demonstrating that rWGS diagnosis rates using CNLP derived descriptions can equal or exceed those obtained using manually compiled ones , 14. ThiAlthough the use of HPO descriptions for WGS-based Mendelian diagnosis is now established practice –10, 14, Our clinical dataset consisted of 293 probands who underwent rWGS at Rady Children’s Hospital in San Diego (RCHSD), 85 of which received a molecular diagnosis of Mendelian disorder. These cases were a sample of convenience drawn from symptomatic children enrolled in previously published studies that examined the diagnostic rate, time to diagnosis, clinical utility, outcomes, and health care utilization of rWGS between 26 July 2016 and 25 September 2018 at RCHSD  , 16, 17.A second independent dataset of 35 probands that were sequenced as part of the University of Utah NeoSeq program  and 2930Highly curated, manually created HPO-based phenotype descriptions were provided for each of the 293 RCHSD and 35 University of Utah WGS cases, as described in NSIGHT1 , 19. Cliscikit-learn, a general-purpose machine learning library written in the Python programming language [2 = 0.0725); accordingly, we used a linear regression to control for this effect. Although one can envision many algorithmic approaches to classification other than Naïve Bayes, e.g., support vector machines or neural nets, for this proof of principle study, we sought to demonstrate feasibility and provide baseline performance metrics. Future work will explore more sophisticated approaches to data modeling.The Mendelian phenotype Search Engine (MPSE) employs Human Phenotype Ontology (HPO)-based descriptions to prioritize patients, determining the likelihood that a Mendelian condition underlies a patient’s phenotype, based upon a training dataset. MPSE does not attempt to determine which Mendelian disease might underlie the patient’s phenotype, rather it seeks to categorize patients as positive or negative for Mendelian disease. MPSE employs a simple, well-established approach: Naïve Bayes . Briefy,language . We alsok = 1) [We validated our results using leave-one-out cross validation  . More spk = 1) .Previous work , 12, 23,MPSE see the “” sectionWe obtained HPO phenotype descriptions for all probands from clinical notes using Clinithink, a third party NLP tool . AutomatComparison of the CNLP descriptions to their corresponding manually compiled ones revealed notable differences with regards to HPO term numbers and contents. The CLiX generated descriptions for the RCHSD and NeoSeq cohorts had an average of 114.8 terms  and 64.5 terms  respectively, whereas the corresponding manually created descriptions averaged 4.1 terms  and 9.5 terms  respectively.t-test (p < 2e−16). The difference in mean MPSE score between Utah sequenced cases (17.3) and unsequenced controls (-33.7) was also statistically different (p = 2e−12). The insert shows a receiver operator characteristic (ROC) curve for the RCHSD data (AUC 0.86), indicating that MPSE can effectively prioritize probands for rWGS. The corresponding AUC for the Utah data was 0.85, essentially identical to the RCHSD result (ROC curve not shown). A possible clinical application scenario can be imagined where MPSE score cutoffs are used to prioritize patients for further review by physicians. For the RCHSD training cohort described here, for example, taking only MPSE scores > 30 would prioritize 30% (89/293) of cases and 4% (31/756) of controls, while taking only MPSE scores > 90 would prioritize 14% (40/293) of cases and 0.8% (6/756) of controls. Anonymized MPSE scores for each patient in these cohorts are tabulated in Additional file We first sought to evaluate how effective our CNLP/MPSE pipeline was at prioritizing patients for WGS. In other words, did the children originally selected for WGS by physicians have higher MPSE scores than those who were not selected? Figure t p < 2e−6. The diMPSE also provides means to identify, and highlight for expert review, those terms in a phenotype description that are most consistent with Mendelian disease. We refer to these terms as the proband’s cardinal phenotypes. Figure To estimate MPSE-driven diagnostic rates, RCHSD and University of Utah sequenced probands were scored using leave-one-out cross validation, as described in the “Figure Both RCHSD and the University of Utah limit manual review of clinical notes to a subset of note types deemed most informative by their institution’s expert reviewers. This is done to speed review by avoiding less informative and redundant note types. A potential advantage of CNLP is that volume is no longer an issue, and every note can be processed. We thus sought to evaluate the utility of processing all notes for every proband. The results of this experiment are also shown in Fig. It is worth noting that underlying NICU populations differ between RCHSD and the University of Utah. Whereas RCHSD is a level IV NICU, the University of Utah operates a level III NICU, with the most severely ill patients transferred to Intermountain’s Primary Children’s neighboring level IV facility. Thus, patients in the Utah dataset are likely to have fewer conditions requiring surgical interventions and a higher level of intensive care. Despite being trained using the RCHSD level IV data, Fig. We have demonstrated the feasibility of prioritizing individuals for WGS, using automated means, and that supplementing clinical review with this automated process could meet or exceed diagnostic yields obtained solely through manual review of clinical notes. More sophisticated machine learning techniques might further improve the accuracy of prioritization. Neural and Bayesian networks and random forest-based approaches generally outperform naïve Bayes. Likewise, addition of other metadata such as provider billing codes, medication histories, ancestry, and socioeconomic indicators might still further improve performance. Nevertheless, even without such enhancements, our CNLP/MPSE workflow prioritized patients for rWGS with relatively high accuracy (AUC = 0.86), with maximal projected diagnostic yields highly enriched for the top scoring quartile. These results bode well for future improved versions of the pipeline.The ability of MPSE to accurately distinguish sequenced from unsequenced probands at both RCHSD and the University of Utah demonstrates the generalizability of the RCHSD training data, at least between two leading research institutions. The fact that MPSE was trained using RCHSD’s level IV NICU patients and replicated in Utah’s level III NICU also provides some indication of MPSE’s robustness and applicability. Broader generalization, however, remains to be proven. Generalization is important because as WGS-based diagnosis becomes more widespread, and patients considered for testing become more diverse, clinical cultures and institutional differences in clinical note taking might render the parameters derived from the RCHSD training dataset less effective at some sites. In this regard, the ability of the pipeline to consume all notes for every proband is clearly an advantage, as it means adopters need not establish cross institutional equivalents in note types; instead, they can simply harvest every available clinical note for every proband.More broadly, generalizability of training data must be distinguished from generalizability of the CNLP/MPSE workflow. The CNLP portion of the pipeline can be used to create a similar dataset for any institution engaged in WGS-based diagnosis, and, because it is a bayesian classifier, retraining MPSE using these data is straightforward. While we chose to use the CLiX CNLP tool, any NLP software able to produce high-fidelity HPO-based phenotype descriptions could be used upstream of MPSE. Going forward, we will explore the utility of retraining and combining models derived from multi-institutional datasets to further improve performance. Recent work has also demonstrated the utility of WGS for pediatric intensive care unit (PICU) patients, where genome-based diagnoses have ended years-long diagnostic odysseys . The PICRe-analysis of previously negative WGS cases is also increasingly an issue. The last decade has witnessed a huge increase in numbers of genes and variants associated with Mendelian conditions , 26, witAdditional file 1: Table S1. Clinical diagnosis frequencies for sequenced RCHSD cases broken down by positive/negative molecular diagnosis status. Table S2. Primary clinical diagnoses for sequenced Utah NeoSeq cases broken down by positive/negative molecular diagnosis status. Table S3. Individual MPSE scores for RCHSD cohort patients. Table S4. Individual MPSE scores for Utah NeoSeq cohort patients.


Preterm birth is defined as babies born alive before 37 weeks of pregnancy or fewer than 259 days since the first day of a woman’s last menstrual period. Globally, 14.84 million babies were preterm births. Preterm infants are at risk for specific diseases related to the immaturity of various organ systems. This study aimed to assess the prevalence of preterm birth and associated factors among mothers who gave birth in public hospitals of east Gojjam zone, Ethiopia.An institutional-based cross-sectional study was conducted from April 1 up to June 30, 2021, in public hospitals in the east Gojjam zone. Systematic random sampling was used. Data were collected through structured questionnaires, patient interviews and patient card reviews. We used binary logistic regression analysis with 95% CI and P-value < 0.05 to identify the significant factors with preterm birth.Out of 615 mothers, 13.2% gave a preterm birth. Antenatal care ), educational status of mother ), husband educational status), Average monthly family income),family size), multifetal gestation , having Premature Rupture Of Membrane ), history of chronic illness ), being HIV positive), Ante-Partum Hemorrhage ), pregnancy Induced Hypertension ), mode of delivery ), and onset of labor ) were found to be significantly associated with preterm birth.antenatal care, educational status of the mother, husband’s educational status, family income, family size, multifetal gestation, Premature Rupture of the membrane, history of chronic illness, being HIV positive, Ante-Partum Hemorrhage, pregnancy Induced Hypertension, mode of delivery, and the onset of labor were found to be significantly associated with preterm birth. To minimize the proportion of preterm birth focusing on this important variables, timely identification of obstetric complications, strengthening early screening of HIV and high-risk pregnancies like multiple gestations, PIH and APH were important. Preterm birth (PTB) is defined as babies born alive before 37 weeks of pregnancy or fewer than 259 days since the first day of a woman’s last menstrual period . GloballPreterm infants have a higher risk of developing specific diseases due to the immaturity of various organ systems and the causes of preterm birth. As a result of their prematurity, preterm babies are subjected to serious illnesses or deaths during their neonatal period. In the absence of appropriate treatment, survivors are more likely to suffer a lifelong disability and a compromised quality of life. Prematurity complications are the leading cause of neonatal death and the second leading cause of death among children under 5 years old. . PrematuIn low- and middle-income (LMIC) countries, preterm births account for more than 60% of all births, and the rate has steadily increased. Despite this high preterm birth rate, it is challenging to determine the trend of preterm birth in the majority of low-income economies due to a lack of accurate data . Sub-SahThe complication of infants born at preterm gestational age results in a trivial cost to the health sector, parents, and society. The prediction and prevention of preterm birth is a major health care priority . Global Previous studies conducted in different regions indicated that several risk factors were identified for preterm birth. This includes having a previous preterm birth, having a short cervix, short intervals between pregnancies, and certain pregnancy complications , chronic illness, educational status, Multiple pregnancies, maternal age, residing in rural areas, antenatal care visits, being HIV positive, family number, and income , 18–26.PTB is a major public health problem. However, in most low-income countries, including Ethiopia, little emphasis is given to PTB intervention as a means of reducing infant mortality. Health care providers or other stakeholders who worked in this public health problem need data related to common factors associated with preterm birth. However, in our country, the studies conducted about this problem were minimal. Although few studies have been conducted in some areas of Ethiopia, the magnitude and possible risk factors of PTB vary by area. A number of methodological issues are addressed in our study  that were not considered in previous studies. Studying a large region is essential to designing effective interventions and programs in public health. Most of the previous study focused mainly on the prevalence of PTB, rather than associated factors of PTB. In addition, different cultures and socioeconomic statuses within a society have varied factors of preterm birth. Therefore studying in different and multicenter settings was important. Determining preterm birth prevalence and associated factors greatly guides health professionals and health policymakers to identify indicators for monitoring preterm birth strategy and applying necessary preventive and appropriate measures to decrease preterm birth. Moreover, the study area’s magnitude of preterm births and associated factors were unknown. Therefore, this study was conducted to assess the prevalence of preterm birth and to identify factors associated with preterm birth in public hospitals in the east Gojjam zone.The hospital based cross-sectional study design was conducted using interviewer administered questionnaire from April 1 up to June 30, 2021. Additional information was obtained from medical records of the mothers and babies.The study was conducted in public hospitals in the east Gojjam zone, Amhara National Regional State, Ethiopia. Debre Markos city is a zonal administrative city. It covers 14,010 square kilometers and is divided into 18 administrative districts, further subdivided into 49 urban and 392 rural kebeles, the smallest administrative units . The EasAll mothers who gave birth in randomly selected public hospitals in the east Gojjam zone during the study period were our study population. Mothers who gave birth and had known either LNMP or had early ultrasound (before 24 weeks) diagnosis were included for this study. Mothers who were deaf and comatose, had unknown last normal menstrual period (LNMP) and had no early ultrasound (before 24 weeks) were excluded from this study.A systematic sampling technique was used. The sample was arranged based on the three-month patient flow before the data collection period by referring to the hospital’s delivery registration book/ record. To calculate K, the summation of the three months delivery report for the hospital was 1698.Then K = N/n, 1698/615 = 2.8 ≈ 3. Where k = interval, N = total population, n = sample size. Every third mother’s was interviewed, gestational age of the newborn was calculated based on the mothers LNMP or first-trimester ultrasound result, in estimating gestational week, when there are extra days it was counted to the near lowest gestational age. The next participant was taken when the selected study participant was not eligible for the study.The sample size was determined by using a single population proportion formula by considering the prevalence (p) of preterm birth = 15.5%, which was obtained from the previous study in Ethiopia , 95% conFinally, after taking a 10% non-response rate the total sample size (n) was 615. An average delivery report for a month before the actual data collecting period was computed for each hospital by analyzing the client’s registration book to distribute the sample size proportionally to each hospital. The sample size was then proportionally allocated to each hospital. Finally, the immediate postnatal mother with her baby had been selected every three intervals using a systematic sampling technique.Data were collected and extracted by reviewing medical records of mother’s .The data collectors were four fourth year undergraduate midwives students from Debre Markos University. One staff midwife from each hospital was assigned to supervise the data collection process. The data collection process was supervised by both the principal investigator and the supervisor. Information collected from the mother included Residency, Age, Religion, Education status, Occupation, Marital status, Average monthly Average monthly family income, Antenatal Care (ANC), inter pregnancy interval parity, Educational Status of husband, Ante-Partum Hemorrhage (APH), Premature Rupture Of Membrane (PROM), Pregnancy Induced Hypertension (PIH), multiple pregnancies, polyhydramnios, anemia, cardiac disease, hypertension, HIV status, Modern contraceptive use before current pregnancy, Urinary Tract Infection (UTI) and malaria were the predictor variable for this study. Anemia was defined as an HGB level below 11gm/dl (HCT < 33%). Obstetric complications was defined as challenges or problems that happen during labor or delivery. PIH was defined clinically as a blood pressure of > 140/90 mmHg after 20 weeks of gestation with or without proteinuria and/or edema as diagnosed and documented by the attending clinician. APH was defined as any vaginal bleeding in the mother after 24 weeks of gestation as documented in the records by the attending clinician. UTI was defined as a documented clinical/laboratory diagnosis of UTI any time during the pregnancy and/or a positive history of treatment of burning sensation with micturition as reported by the mother. Birth to pregnancy interval was defined as the time between the start of the index pregnancy and the preceding live birth. Last normal menstrual period was defined as the date of the starting of last normal menstruation the women had to index pregnancy. Preterm birth was defined as a newborn with a gestational age of 28 weeks to less than 37 weeks. To assure the quality of data, the questionnaire was pre-testing on 31 mothers in public hospitals in the east Gojjam zone, and the questionnaire’s fitness was confirmed from pre-testing. One-day practical training on how to collect data was given to the data collectors and the supervisor before data collection. During the data collection period, the collected data were reviewed, checked for completeness, and signed by the supervisor at the end of each day.All questionnaires were checked, coded, and entered into the SPSS version 25 software packages, which were then analyzed using R 4.1.3. The data were presented using frequency tables and graphs. The relevant determinants of preterm birth were identified using binary logistic regression. The researchers used both bivariable and multivariable analyses. In the bivariable analysis, independent variables with a p-value less than 0.25 were chosen for the multivariable analysis. An adjusted odds ratio with a 95% confidence level was used to examine the degree of relationship between independent and dependent variables, and variables with a p-value of 0.05 were considered statistically significant.All participants completed the interview (100% response rate). The majority of the respondents 358 (58.2%) were rural residents. The majority of the study participants 360 (58.5%) were between 25 and 34 years old. The entire respondent belongs to Amhara by ethnicity, and 516 (96.1%) were Orthodox Christians in religion. Regarding the marital status of the respondents, the majority of them 596(96.9%) were married. 253 (41.1%) were have no formal education, 217(35.3%) of the respondents were taken education in primary school, and 253(41.1%) of them can only read and write, and the remaining 145 (23.6%) were secondary and above education level. The occupational status of most of the respondents 228(37.1%) were farmers, 144 (23.4%) of the respondents were housewives, 150 (24.4%) of the respondents were employers, and the remaining respondents had other occupations . 264 (42.9%) of the husband were have no formal education. 264(42.9%) of the respondent’s family average monthly income is less than or equal to 3000 Birr and 212(34.5%) of the respondent’s family average monthly income is average greater than 5000 Birr. The majority of the respondents, 415 (67.5%) had between 3 and 5 family members (Table Most of the respondents (96.3%) had ANC follow up and 56.1% of the respondent had at least 4 visits. The majority of the respondents used modern contraceptives (94.8%) before their pregnancy, and the majority of mothers had their pregnancy wanted and planned (99.3%). More than two-thirds of the respondents had birth-to-pregnancy intervals greater than or equal to 36 months (83.5%). Labor spontaneously started in 87.8% of the respondents. Three-quarters of the respondents (74.3%) gave birth by SVD. Out of the total respondents, 595(96.7%) were tested for HIV status, and 36 (5.7%) respondents were HIV positive. (133) 21.6% of women had APH, (142) 23.1%, of women had PIH, (70) 11.4%, of mothers had multiple pregnancies, and women had Polyhydramnios (10) 1.6%. 105(17.1%) of the respondents had a history of chronic illness. 70(11.4%) of mothers had urinary tract infection  ), educational status of mother ), husband educational status), Average monthly family income),family size), multifetal gestation , having PROM), history of chronic illness ), being HIV positive), APH), PIH ), mode of delivery ), and onset of labor ) were found to be statistically significant at p-value of < 0.05 (Table .75),famiThis study was conducted to assess the magnitude of preterm birth and its associated factors in public hospitals in the east Gojjam zone. During the study period, the overall proportion of preterm birth was found to be 13.2%. The finding was greater than the preterm birth rate for the world (9.8%) and North America (9%) . It was The proportion of preterm birth in this study however was higher than another study conducted in our country at Gondar town health institutions, which reported a prevalence rate of 4.4% . This diThe study conducted in Malawi shows that the prevalence of preterm birth was 16.3% . This hiThe current study’s finding was lower than those conducted in Kenya National Hospital and Jemma University Specialized Hospital, which reported the prevalence of preterm birth was 20.2% and 25.9%, respectively , 38. ThiMothers with less than 3000 birr Average monthly family income were 1.95 times more likely to develop preterm than those with greater than 5000 birr family income. This study was in line with the study in SSA , 39,whicMothers from three up to five family members were 0.15 times less likely to develop/give preterm as compared to mothers from greater than five family members. This study was in line with the study in Ethiopia , which sThe education status of mothers were significant factors of preterm. Mothers who had no formal education were 2.79 times more likely to develop/give preterm than mothers who had secondary and above education levels. This study was in line with the studies in Sub-Saharan African and European countries –41, whicMothers who had husbands who had no formal education were 2.11 times more likely to develop/give preterm than mothers who had husbands with secondary and above education levels. This study is in line with the previous study in Iran , which sMothers with APH were 3.62 times more likely to have a preterm birth than mothers without APH. This finding is in line with the study conducted in Kenya, Ethiopia, and East Africa , 44, 45.Mothers with multifetal gestation were 3.30 times more likely to have a preterm birth than mothers without multifetal gestation. This finding is in line with the study conducted in Ethiopia, Korea, Greek –48. It mMothers who delivered with SVD were 7.16 times more likely to develop preterm as compared to mothers delivered with CS or Instrumental delivery. This finding is in line with the study conducted in Ethiopia , which sMothers with spontaneous labor were 0.10 times less likely to have preterm birth as compared to mothers with induced labor. This finding is in line with the study conducted in Ethiopia , which sHistory of chronic illness was significantly associated with the outcome variable, mothers who had a history of chronic illness were 3.94 times more likely to give preterm birth than mothers who had no chronic illness history. This finding is in line with a study conducted in Ethiopia , 44, 49,This study revealed a significant association between pregnancy-induced hypertension and preterm birth. Mothers who had complications PIH were 3.61 times increased risk of having a preterm birth than those mothers without this problem during the index pregnancy. This finding is in line with the study conducted in East Africa, Ethiopia, Nigeria, Iran, Ghana, and Kenya , 51–55, Another significant association was found between mothers who had premature rupture of membranes (PROM) and preterm birth. Mothers with PROM were 6.46 times more likely to have a preterm birth than their counterparts. This finding was in line with the previous findings in Tehran, Iran, Ethiopia, East Africa, Nigeria , 56, shoIn this study, the ANC follow up were significantly associated with the outcome variable preterm birth. Mothers with < 4 times ANC follow-up in the index pregnancy were 2.87 times more likely to have a preterm birth than mothers who had the ANC visits ≥ 4 times. This finding is in line with a study conducted in Ghana, Nigeria teaching hospitals, and western Ethiopia , 54, 57,Being HIV positive was significantly associated with preterm birth. HIV-positive mothers were 6.99 times more likely to give preterm compared to HIV-negative mothers. This finding is in line with a study in Malawi and Ethiopia , 58, 59,In contradiction to the previous studies in our study, age, marital status, RH factor, Pregnancy status, and UTI , 62, 63.Despite efforts to reduce recollection biases by informing local events, there may be a recall bias. When ultrasound facilities are not accessible, the menstrual history and clinical examination are used to confirm gestational age and which may subject to considerable error. Another drawback of our study is using secondary data for some variables.The proportion of preterm birth in public hospitals in the east Gojjam zone is 13.7%. number of ANC, educational status of mother, husband educational status, family income, family size, occupation, multifetal gestation, having PROM, history of chronic illness, being HIV positive, APH, PIH, mode of delivery, and onset of labor were found to be significantly associated with preterm birth. As a result, focusing on these important variables would reduce the number of premature births. Furthermore, it was suggested that educating the community about the importance of ANC service utilization of mothers and preventing preterm birth be strengthened. Moreover, strengthening early screening of HIV and high-risk pregnancies like multiple gestations, PIH and APH were important to prevent preterm.


Ossa cordis, and os aorta using computed tomography soft tissue window (CT) alongside 3D render volume reconstructions and light microscopy. The current study techniques demonstrated the Ossa cordis and os aorta in the cardiac window with more precision than the black and white (ghost), and angiography images. Transverse and sagittal CT images additionally demonstrated the presence of Ossa cordis and os aorta. This study is the first to record two small Ossa cordis sinistrum and one os aorta in the camel heart, in addition to the more commonly observed singular, large, os cordis dextrum. The os cordis dextrum was always located in the upper part of the interventricular septum, near to its junction with the atrium, forming an elongated rectangular shape when observed transversally. The wider cranial part was composed from bone, whereas the caudal aspect was narrow and contained both bone and cartilage. Light microscopy identified that the os cordis dextrum consisted of trabecular bone, marrow spaces, and hyaline cartilage. Two Ossa cordis sinistrum were detected on the left side of the heart, one in the right fibrous ring and another in the interventricular septum, microscopy showed that both contained only trabecular bone with osteocytes, osteoblasts, and osteoclasts. At the level of ascending aorta, there was also trabecular bone containing osteocytes, an os aorta.The present study describes the morphological characteristics of the camel heart  Ossa cordis and os aorta in the camel. Ossa cordis sinistrum in the camel.Discovery of os cordis dextrum and two Ossa cordis in camels.Discovery of os aorta in addition to Ossa cordis and os aorta.Combined use of computed tomography and light microscopy to analyze 1Camel dromedarius) is extremely well adapted to life in hot and arid lands. Dromedary camels have very special anatomical and physiological characteristics, which enables these animals to live, reproduce, produce milk, and meat, and to thrive under these extreme heat and arid conditions, even during times of drought when cattle, sheep, and goats may fail to survive  and Lamini (New World camels) tribes diverged around 16.3 million years ago  contains four rings of dense fibrous connective tissue into which heart muscle fibers are inserted, helping provide structure for the heart alongside isolating electrical signaling between the ventricles and atria  between the atrioventricular openings and therefore the aortic opening. Previous work in differing species has shown that Ossa cordis dextrum are more common, and are usually larger, than those on the left  cattle, sheep, goats, pigs, otters and chimpanzees  using CT, 3D CT and histological examinations to provide a detailed study on Ossa cordis. The present work also discovered that camels can have not just a singular os cordis dextrum, as previously stated, but could also have two smaller Ossa cordis sinistrum and one os aorta.Despite these earlier reports describing the basic morphology of the heart and the histology of the single os cordis in the dromedary camel, studies have not been conducted using detailed imaging such as CT or 3D CT. Therefore, this study investiagted 2Hearts from five adult, male, healthy camels aged 13 years old, with no history of clinical cardiac abnormalities were slaughtered by a professional veterinary surgeon. The camels were not slaughtered for research purposes, permission was granted to work on the hearts. The work was approved by the Animal Welfare and Ethics Committees, in the Faculty of Veterinary Medicine, Alexandria University and the School of Veterinary Medicine and Science, University of Nottingham , in accordance with institutional, national and international guidelines. The hearts were dissected out at the slaughterhouse, placed on ice, and immediately transferred to the laboratory. The hearts were examined and imaged within 2 hours of death in order to reduce any potential postmortem variations. Immediately following CT imaging, the specimens were processed for histology.2.12, using a continuous helical scan mindose technique. After obtaining a preliminary image to determine the 3D‐CTA scan range, serial cross section scans from the apex to the base of each heart were conducted using a slide thickness of 1 mm with 1.3 mm intervals , with the following soft window settings: mAs 240, Kv 130, W.342 L.52.Each camel heart was positioned on its right surface for each scan. The hearts were wrapped in a secure in foam material to avoid any movement that would interfere with image quality. The CT X‐ray examinations were performed on an AMDCT scanner with 128 detectors , with a rotation time of 300 ms, and a slice collimation of 128 × 0.6 mmTo obtain sagittal sections using the soft tissue window, the scans were conducted at the level of the interventricular septum and about 20 scans were made with 1.8 mm intervals at a thickness of 8 mm, with setting set at: mAs 240, Kv 130, W.342 L.52. CT image reconstruction was undertaken using the optimal reconstruction parameters for hearts. Initially, a set of cross‐sectional tomography slices were constructed individually, then the images were stacked together sequentially to obtain a 3D image model of each heart. The reconstruction algorithm was used within octopus software, then converted into a DICOM format producing three reconstruction images: ghost (black and white) render, color render (red in color) and an angiograph which was translucent blue  to measure the 2.2Ossa cordis dextrum and sinistrum and os aorta, as observed from the CT scans, were processed for histological analysis. These samples were then fixed in 10% buffered neural formalin and decalcified. The samples were decalcified using 10% ethylene damine tetra acetic acid (EDTA) in 0.1 M Tris/HCl buffer, pH 7.4, for 6 days, with solution changes every 2 days  and llama (Lama glama), which also resides in the Artiodactyla order, Tylopoda suborder, within the Camelidae family  was present in association with Ossa cordis dextrum. The presence of trabecular bone alongside cartilage was also reported in camels previously . Two other hearts  showed a single os cordis dextrum. The os cordis dextrum was located in the upper part interventricular septum, near its junction with the atrium, and passed transversely through the structure in an elongated rectangular shape. The cranial part of the os cordis dextrum was wider and ossified, whereas the narrow caudal part contained both trabecular bone and cartilaginous tissue. The smaller Ossa cordis sinistrum were located in different locations, with one within the right fibrous ring and the other in the interventricular septum. The os aorta was in the wall of the ascending aorta. This research represents the first time a camel heart has used CT scanning and histology to investigate Ossa cordis. The presence of an os cordis dextrum, Ossa cordis sinistrum and os aorta within the camel is a novel discovery within any species.A novel discovery of one os cordis dextrum, two Samir A.A. El‐Gendy: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; validation; visualization; writing – original draft; writing – review and editing. Mohamed A.M. Alsafy: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; validation; visualization; writing – original draft; writing – review and editing. Catrin S. Rutland: Conceptualization; formal analysis; funding acquisition; investigation; validation; visualization; writing – original draft; writing – review and editing. Samar M. Ez Elarab: Data curation; formal analysis; investigation; methodology; validation; visualization; writing – original draft; writing – review and editing. Hanan H. Abd‐Elhafeez: Data curation; formal analysis; investigation; methodology; validation; visualization; writing – original draft; writing – review and editing. Basma Kamal: Data curation; formal analysis; investigation; methodology; validation; visualization; writing – original draft; writing – review and editing.The authors would like to thank the Faculty of Veterinary Medicine, Alexandria University and the School of Veterinary Medicine and Science, University of Nottingham.None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.