Whole genome duplication (WGD) is a dramatic evolutionary event generating many new genes and which may play a role in survival through mass extinctions. Paddlefish and sturgeon are sister lineages that both show genomic evidence for ancient WGD. Until now this has been interpreted as two independent WGD events due to a preponderance of duplicate genes with independent histories. Here we show that although there is indeed a plurality of apparently independent gene duplications, these derive from a shared genome duplication event occurring well over 200 million years ago, likely close to the Permian-Triassic mass extinction period. This was followed by a prolonged process of reversion to stable diploid inheritance (rediploidization), that may have promoted survival during the Triassic-Jurassic mass extinction. We show that the sharing of this WGD is masked by the fact that paddlefish and sturgeon lineage divergence occurred before rediploidization had proceeded even half-way. Thus, for most genes the resolution to diploidy was lineage-specific. Because genes are only truly duplicated once diploid inheritance is established, the paddlefish and sturgeon genomes are thus a mosaic of shared and non-shared gene duplications resulting from a shared genome duplication event. Whole genome duplication can generate new genes and support survival through mass extinctions. Here, the authors show that paddlefish and sturgeon shared a genome duplication event 200 million years ago that was previously unrecognised due to the mixed signals from independent rediploidisation. These events are often hypothesised to have facilitated evolutionary success through provision of the raw genetic materials for phenotypic innovation and species diversification23. A key evolutionary process after WGD is rediploidization—the transition of a polyploid, usually tetraploid, genome to a more stable diploid state25. Importantly in this context, WGD events are derived from either hybridisation of two different parent species  or from doubling of the same genome at the intra-species/individual level (autopolyploidisation), each with distinct cytogenetic outcomes. Classically, the non-homologous chromosomes of new allopolyploids preferentially pair bivalently during meiosis, whereas the four homologous chromosomes of new autopolyploids take a multivalent formation24. This results in ongoing homologous recombination, and hence gene conversion and homogenisation, across the four allelic copies at each locus. Suppression of recombination, probably achieved through chromosomal rearrangements and other mutations, is a necessary step to rediploidize these genes into two distinct  ohnolog loci (WGD-derived duplicate genes) from tetraploid alleles. It is only then that uninterrupted sequence and functional divergence can occur between ohnolog pairs24. The rediploidization process thus uncouples the genome duplication process from the gene duplication process in autopolyploids, as it is only once rediploidization has occurred that the locus can be considered duplicated.Ancient WGD events have occurred across the tree of life and are especially well studied in plants26, and ancestral teleost WGD28, indicates that autopolyploid rediploidization can be temporally protracted, occurring asynchronously across the genome over tens of millions of years28. Major implications arise from the accompanying delay in any evolutionary processes that depend on ohnolog genetic divergence24. These include well-established models of functional evolution after gene duplication, e.g. sub-/neo-functionalization30, and models of reproductive isolation involving reciprocal loss of ohnologs in sister lineages31. Furthermore, if speciation occurs before rediploidization has completed in descendent lineages of the same WGD, rediploidization may occur independently in these daughter lineages (lineage-specific rediploidization) in some genomic regions24. This in turn allows for ohnolog pairs to independently evolve divergent regulatory and functional trajectories in each lineage, potentially in response to lineage-specific selective pressures24. Ohnologs with this history have been described as following the LORe (Lineage-specific Ohnolog Resolution) model24.Substantial evidence arising from studies of the ancestral salmonid WGD34, it remains unclear whether asynchronous and lineage-specific rediploidization is a general feature after autopolyploid WGD outside the teleost clade. The past few years have seen the generation of high-quality reference genomes from multiple non-teleost ray-finned fish lineages36, which share the two ancient rounds of WGD common to all jawed vertebrates37, but lack the teleost-specific WGD event39. These species typically have slowly evolving genomes, enabling more reliable inference of the ancestral state of bony vertebrates than teleosts, while providing outgroups to understand the impact of the teleost-specific WGD event39. Among these newly available genomes are chromosome-scale assemblies for the sterlet sturgeon (Acipenser ruthenus)7 and American paddlefish (Polyodon spathula)8, which each have experienced WGD in their evolutionary histories.Although noted as a potentially important evolutionary process after several WGDs42. On the other hand, American paddlefish is the only extant paddlefish43, and is suggested to have undergone a single WGD event46. Despite being sister lineages, together representing extant Acipenseriformes, previous analyses have consistently rejected a shared ancestral WGD in favour of independent WGD events  to mask a shared WGD event has never been formally proposed or tested in any lineage.Multiple WGD events have occurred during sturgeon evolution, but only one, thought to be shared by all sturgeons, is present in the sterlet sturgeon’s historynts Fig. 8,35,38,4Here, adopting a phylogenomic approach, we reconsider the timing of WGD(s) relative to the sturgeon-paddlefish divergence, accounting for the possibility of lineage-specific rediploidization after a shared WGD. Taking care to distinguish our results from phylogenetic error, and drawing on conserved synteny, we provide strong evidence for a single ancestral autopolyploidy occurring close to the Permian-Triassic extinction event. This was followed by extensive lineage-specific rediploidization, resulting from the ancestral acipenseriform genome remaining predominantly tetraploid at the time of sturgeon-paddlefish divergence, a genomic condition that may have promoted survival of the lineage through the Triassic-Jurassic mass extinction.44. This approach considers two scenarios as plausible: (1) if a plurality of ohnolog gene trees recover independent duplication nodes after the sturgeon-paddlefish divergence then sturgeons and paddlefish are assumed to have undergone independent WGDs (currently accepted hypothesis)  a shared WGD followed by a prolonged rediploidization process that starts before but continues after speciation. This predicts the presence of two distinct subsets of ohnolog gene trees, one with duplication nodes prior to the sturgeon-paddlefish divergence and the other with independent duplication nodes after speciation  in acipenseriform history have sought a single consensus ohnolog divergence time relative to speciationausible:  if a pluausible:  if a pluausible:  if a plu7, integrating extensive additional phylogenetic and syntenic evidence. Specifically, we incorporated a broad sampling of predicted proteomes from jawed vertebrate genomes, including from newly available non-teleost ray-finned fish. This allowed us to define 5,439 protein-coding gene families containing high confidence ohnolog pairs in both sturgeon and paddlefish. Analysing maximum likelihood gene trees for each family we found that the gene tree harbouring independent duplication nodes was the most common topology , being recovered 2074 times . First, we considered the recovery of three broad topology categories , and two PreSpec-type  -testske) Fig. . The reske) Fig. . Althougke) Fig. . Rather 48) within the sturgeon-paddlefish ohnolog pair subtree. As stringency increases, we observe a drop out of all tree topologies  than PostSpec or PreSpec topologies  within the sturgeon-paddlefish ohnolog pair subclade never resulted in a topology change, while support values never dropped below UFBoot = 97% 57.Among these apparently single-copy paddlefish genes, there is a greater proportion of PostSpec-type trees  to PreSpec-type  than recovered for the main data above .If a small proportion of duplicated paddlefish loci are still undergoing tetrasomic inheritance or are artefactually collapsed into a single locus, then this should be apparent as regions with twice the sequencing depth compared to the rest of the genome. This is because reads derived from two distinct genomic locations should map to collapsed regionsThe main density peak when plotting per-gene sequencing read depth for the single-copy gene sets is comparable to each of the two-copy paddlefish ohnolog pair genes. This implies that gene loss in paddlefish, rather than maintenance of tetraploidy or assembly collapse, explains most of these cases   between paralogous genes within a genome is often applied as the basis for detecting WGD events and estimating their timing relative to speciation61. This is typically achieved through statistical modelling and/or simple visual identification of distribution peaks in Ks plots.The distribution of synonymous substitutions per site  rediploidization has occurred it naturally follows that a strong, single Ks values for species-specific ohnolog pairs from the PreSpec, PostSpec, and PreSpec- and PostSpec-like datasets, as well as all these combined , and lower Ks of late rediploidizing ohnologs (i.e. PostSpec), compared to ortholog Ks values is consistent with our predictions, and lends further support to the scenario of a shared WGD followed by an asynchronous rediploidization process.With this in mind, and to further explore the differences between our PreSpec, PostSpec, and ‘Other’ topology datasets, we calculated pairwise ese Fig. . This hiKs peaks also fall intermediate to the PostSpec and PreSpec pair Ks peaks, and on a gradient with PostSpec-like always lying closer to PostSpec and PreSpec-like always closer to PreSpec  are consistently recovered for paddlefish than for sturgeon , this likely explains the trend towards higher Ks values in paddlefish.Ohnolog pair eon Fig. . This maeon Fig. . This suKs peak may be compromised by protracted rediploidization periods.In all, these results are clearly consistent with asynchronous rediploidization masking a shared ancestral WGD in the sturgeon-paddlefish ancestor. Moreover, they indicate that approaches reliant on identifying and dating of WGD events by detecting a single 62. Our gene tree and Ks analyses suggest this is likely a major factor influencing the disparate array of dates previously proposed for WGD in sturgeon and paddlefish. Although imperfect, the most reliable lower bound estimate for the ancestral sturgeon-paddlefish WGD event can be estimated from ohnolog pairs that rediploidized prior to the sturgeon-paddlefish divergence, as these will have diverged closer in time to the WGD event62. With this in mind, to estimate a lower bound timing for the WGD we took a Bayesian phylogenomic approach63 using concatenated ohnolog pairs62 based on the set of 81 gene trees that maximally supported shared WGD and did not include duplicates in other species. We analysed five distinct datasets, always including all 81 gene families but randomly shuffling ohnologs from a pair for arbitrary assignment as the A or B copy for concatenation, to avoid bias and assess robustness of results to alternative concatenations62. We analysed these datasets with an autocorrelated relaxed molecular clock64, and used the site-heterogeneous CAT-GTR substitution model65, and considered two fossil calibration strategies  we infer a divergence time of ~171.6 Ma  for the split of sturgeons and paddlefish    appear to show particularly strong chromosomal homology between sturgeon and paddlefish , will also have been ancestral to both extant acipenseriform lineages. This partially shared rediploidization history after shared WGD likely explains at least some of the proposed similarity in genome evolution between paddlefish and sturgeons and perhaps contributes to their remarkable ability to hybridise67.Though not framed in the context of a shared WGD, similarity in the evolution of the sturgeon and paddlefish genomes has been previously notedish Fig. 8; and we24. Given that over half of the genome appears to have rediploidized after the sturgeon-paddlefish divergence, it is likely that these independently rediploidized ohnologs, and the networks they form, contribute substantially to the unique biology of each lineage. For example, a recent study of the oxytocin and vasotocin receptor (OTR/VTR) gene family, which play a variety of roles, including in social behaviour and reproduction, found that these duplicated genes emerged consistent with independent WGDs in each lineage68. Our results instead indicate that genes from the OTR/VTR family might be better interpreted as following the LORe model24, having rediploidized independently in sturgeons and paddlefish.Conversely, other parts of the genome—those chromosomes and regions of chromosomes that were tetraploid when sturgeons and paddlefish diverged— rediploidized independently in each lineage, with different (and differently sized) regions, and hence different sets of genes, rediploidizing at different times. Those ohnologs that resolved from alleles after the speciation must have also undergone any sub-/neo-functionalisation/regulation independently26) and teleosts (rediploidization largely resolved after ~60 million years69)  in sturgeons and paddlefish contributed to a more prolonged rediploidization period in Acipenseriformes compared to faster evolving teleosts. This may also help explain the apparent collapse of some ohnologous regions in the paddlefish genome into a single assembly sequence, although we cannot definitively exclude maintenance of tetraploidy for a very small proportion of the genome57. Despite this slower rate of genome rearrangement and rediploidization, and the presence of large blocks of genes sharing consistent rediploidization history in our analyses, we find ohnolog blocks that diverged before and after speciation on the same chromosomes, similar to observations in salmonids26. This appears to have often occurred through temporally isolated intrachromosomal rearrangement events, perhaps facilitating suppression of homologous recombination, and allowing resolution of ohnologs from alleles for that genomic segment. This scenario is akin to the stepwise formation of evolutionary strata on mammalian sex chromosomes58, and adds a layer of complexity atop the existing model of segmental rediploidization proposed for sturgeon7.Our estimate that at least 50–66% of the duplicated genome remained tetraploid at the point of the sturgeon-paddlefish divergence i.e. ~80 million years post-WGD, is much more drawn-out than equivalent estimates for salmonids 70, which is dated at a minimum of ~121 Ma72, this hard minimum directly contradicts all previous estimates with the exception of ~180 Ma based off the sturgeon genome analysis7. Furthermore, rediploidization occurring asynchronously means that common approaches  used to directly estimate the absolute date of autopolyploid WGD events are unavoidably problematic, as they conflate ohnolog rediploidization time(s) with the WGD itself62. This means that all ohnolog pairs likely bias dating towards the present, unless they rediploidize near instantaneously after WGD, but this will be most problematic for ohnologs diverging after the sturgeon-paddlefish divergence (PostSpec). As previous studies did not consider asynchronous rediploidization, all past dating analyses will have incorporated these PostSpec ohnolog pairs in their estimation of WGD timing. We excluded these ohnolog pairs from our analyses and, unlike previous studies, estimated the WGD lower bound using a sophisticated phylogenomics approach. Thus, although our estimated WGD lower bound timing is far older than previous estimates, it should also be more accurate. Further, because of asynchronous rediploidization, it is likely still an underestimate of the true WGD timing.Our WGD lower-bound timing  is substantially older than all previous estimates73. Our dating of the sturgeon-paddlefish WGD lower bound suggests that the event may have occurred close to the P-Tr mass extinction period, yet our mean timing depends on calibration strategy, and confidence intervals are wide.Interestingly, our results support a potential role for polyploidy and asynchronous rediploidization in Acipenseriformes surviving the P-Tr and/or Tr-J mass extinction event(s). It has been proposed that WGDs in plants may confer tolerance and adaptability to extreme environmental conditions, increasing fitness in the face of mass extinction events77 likely split from the ancestor of extant Acipenseriformes with genomes still early in the rediploidization process.However, considering that most rediploidization post-dates the sturgeon-paddlefish divergence, our estimates for the WGD lower bound and of the sturgeon-paddlefish divergence are fully consistent with a model where the flexibility and functional redundancy intrinsic to a genome in the early stages of autopolyploid rediploidization contributed to the survival and success of the Acipenseriformes through at least the Tr-J mass extinction, if not also the P-Tr extinction. However, a duplicated genome is not a general shield against all extinction, and our older WGD lower bound estimates suggest it may have occurred relatively early in chondrostean evolution, implying that stem Acipenseriformes (such as †Peipiaosteidae and †Chondrosteidae)Ks plots and leading to some duplicate genomic regions being so similar as to be collapsed into a single assembly region, or perhaps even still tetraploid, long after WGD (well over 200 million years after WGD in paddlefish). In this context, through identifying distinct signals for lineage-specific rediploidization , we provide a path forward for detecting and analysing other WGD events affected by asynchronous, lineage-specific rediploidization. We note that our framework to distinguish the gene tree distribution signal for lineage-specific rediploidization from that of phylogenetic error does not rule out alternative biological phenomena (incomplete lineage sorting or hybridisation78) as the source of the conflicting gene tree topology distribution. However, even the simplest scenarios required to implicate these factors rather than asynchronous rediploidization are far less parsimonious 8 ohnologs to this dataset we used OrthoFinder82 (v 2.5.4) to infer phylogenetic hierarchical orthogroups (PHOGs). For OrthoFinder analyses we also included a set of proteomes from species spanning jawed vertebrate phylogeny, including the ghost shark 83 and whale shark 84 from Chondrichthyes, and human , domestic chicken , African clawed frog , and coelacanth  from Sarcopterygii. Within Actinopterygii we selected zebrafish , fugu , spotted gar 39, and bowfin 35 as representatives of Neopterygii, the sister group to sturgeons and paddlefishes, and grey bichir 35 as their combined sister group. The longest protein sequence for each gene was used for these analyses where alternative transcripts were annotated. Default OrthoFinder settings were used, with two exceptions. First, we specified a species tree, in line with accepted jawed vertebrate relationships85, to augment orthology inference: “,)),,,)))));”. Second, the OrthoFinder -y flag was specified to further split PHOGs that underwent duplications after the jawed vertebrate ancestor into separate PHOGs. In post-processing of OrthoFinder results, we then performed an extra check by extracting only PHOGs including sequences from as many species as possible, while always including two sequences each from sturgeon and paddlefish. This was achieved by extracting the set of sequences descended from the most ancient ancestral species node in the OrthoFinder reconciled gene tree to not include additional sturgeon or paddlefish sequences . As a simple example, in an orthogroup with a gene duplication in the ancestor of Actinopterygii, with both paddlefish and sturgeon ohnologs being retained in both actinopterygian duplicates, our approach results in two resultant PHOGs, split at the level of Actinopterygii, with neither including sequences from the other duplicate or their co-orthologs from Sarcopterygii or Chondrichthyes, and both containing two sturgeon and two paddlefish sequences each.In their analysis of the sterlet sturgeon genome, Du et al.7, as well as those including at least one outgroup to allow rooting of the sturgeon-paddlefish ohnolog pair subtree, and hence inference of duplication node time relative to speciation. We also excluded gene families where both ohnologs in either paddlefish or sturgeon were present on the same chromosome, or where any paddlefish or sturgeon sequence was present on a scaffold not assigned to one of the 60 sturgeon or paddlefish chromosomes. Lastly, we checked that the sturgeon and paddlefish sequences formed a monophyletic group in inferred PHOG gene trees (see below section on Ohnolog duplication time inference). 5439 PHOGs met these criteria (and form our ohnolog-pair set), of which 5372 also included at least one sequence each from Neopterygii and a more distantly related outgroup, all but ensuring that the two paddlefish and sturgeon sequences diverged after splitting from Neopterygii.These PHOGs were then filtered to retain only those that matched a previously inferred sturgeon high-confidence ohnolog pairIn all, this should have resulted in a dataset heavily enriched for ohnolog pairs in sturgeon and paddlefish. However, we note that the set includes only ohnolog pairs where both ohnologs are retained in both species and excludes PHOGs that have undergone additional duplications or losses in sturgeons, paddlefish, or their ancestral stem lineage. Similarly, it is possible that a very small subset of our ohnolog pairs may derive from complex scenarios of multiple ohnolog losses and inter-chromosomal duplications that have evaded our filters  and left a pair of ohnolog look-a-likes in paddlefish and sturgeon. However, we expect PHOGs where this has occurred to be exceedingly rare in our final 5439 sturgeon-paddlefish ohnolog-pair dataset.86 (v. 2.1.4-beta COVID-edition), with the -m JTT + G flag to use the JTT87 amino acid substitution model with four discrete gamma categories, as well as the -bb 1000 flag to specify 1000 ultrafast bootstrap replicates48. The resulting maximum likelihood trees were extracted for pre-processing and duplication time inference. To pre-process these trees, we used the ETE (v3) toolkit88 python library to check that sturgeon and paddlefish sequences formed a monophyletic (in an unrooted sense) clan89 in each PHOG gene tree, and then rooted each tree with the most distantly related sequence relative to sturgeons and paddlefish . In a separate python script, the ETE toolkit was then used to perform strict gene tree-species tree reconciliation90 to infer speciation and duplication nodes/events, before classifying  and summarising the different sturgeon-paddlefish subtree gene tree topologies and frequencies recovered.To estimate rediploidization time (i.e. duplication node time) relative to speciation for each sturgeon-paddlefish ohnolog-pair we performed phylogenetic analyses for each gene family from the pre-monophyly filtered sturgeon-paddlefish ohnolog pair PHOGs described above (5590 trees). Multiple sequence alignments were performed with MAFFT v7.487 with the --auto flag specified, and the --anysymbol flag for those datasets that included sequences containing selenocysteine (symbol U). Phylogenetic inference was performed using IQ-tree47 analyses in IQ-tree86. To do this we classified the three possible unrooted topologies of our four-tipped sturgeon-paddlefish subtree as either PostSpec-type or PreSpec-type based on whether they would become PostSpec(-like) or PreSpec(-like) if rooted. For each of the four rooted topology categories  we extracted the subalignment for the four sturgeon/paddlefish sequences from each PHOG in that category, and then performed an AU-test analysis for each ohnolog-pair set specifying the three possible unrooted topologies. The frequency at which the matched unrooted topology type was not rejected for subalignments from each rooted tree topology category was then calculated and plotted.To interrogate the possibility that either of the PreSpec or PostSpec gene trees derive from error, as well as to better understand the source of the ‘Other’ topologies we performed a suite of analyses. First, we performed unrooted AU-test88 we assessed the influence that filtering our ohnolog-pair PHOG counts by increasingly higher ultrafast bootstrap percentage cut-offs  would have on rooted topology frequencies. Starting at a cut-off of 0% and incrementing by 5%, up to 100%, we assessed the total counts and percentages of PostSpec, PreSpec, and ‘Other’ tree topologies recovered at each cut-off and then assessed for trends in topology frequency as the cut-off became more stringent. We also calculated and plotted the fold deviation from random  at which each rooted tree category was recovered for PostSpec (randomly expected 1/15 times), PreSpec (randomly expected 2/15 times) and ‘Other’ (randomly expected 12/15 times) topologies across the same series of ultrafast bootstrap cut-offs and evaluated the trends observed.Next, using a python script and the ETE toolkit50, and hence may be more likely to be misleading, we used a custom ETE toolkit88 python script to assess for the monophyly of three widely-accepted clades; Tetrapoda , Teleostei , and Chondrichthyes . This script also required that at least one sequence was present for each species in that monophyletic clade, meaning some negatives may also derive from gene loss or absence from our inferred PHOG.To assess whether gene families supporting any topology perform poorly at recovering other generally-accepted clades91. Alignment length and average pairwise percentage identity were calculated using the esl-alistat program from the Hmmer package 92, while the number of parsimony informative sites52 was extracted from IQ-tree output. PhyKIT (v. 1.11.3)51 was used to compute the number of variable sites52, the evolutionary rate 53, treeness 54, relative compositional variability (RCV)54, treeness/RCV54, and saturation level55.To detect signs of possible systematic errors we compared a range of statistics at the sequence alignment, modelling, and inferred phylogenetic tree levels, between gene trees fitting the PostSpec, PreSpec, and ‘Other’ tree topologies using the two-sided Wilcox-test with Bonferroni correction in R (version 4.1.2 [2021-11-01])93, EX_EHO94, and JTT + C2095 models, which can help to alleviate systematic biases, and often fit single gene family alignments well97.To further explore whether our results could derive from systematic error, we tested the use of precomputed site-heterogeneous mixture models on the set of PHOGs that had maximal support for any sturgeon-paddlefish subclade topology . Specifically we tested the fit and influence the UL3https://db.cngb.org/search/project/CNP0000867/)8 to the paddlefish genome assembly using Bowtie (v. 2.4.2). A sorted BAM file was generated for the aligned data using SAMtools (v. 1.16.1)98. For three separate gene sets  gene coverage was calculated by extracting raw read depth per gene  from the BAM using the --by flag in mosdepth (v. 0.3.3)99.We analysed the set of ohnologs where both genes of the pair were present in sturgeon and only a single gene was present in paddlefish. This was performed by closely following the approach in the Ohnolog-pair datasets and Ohnolog duplication time inference sections above, but this time choosing PHOGs that contain only a single paddlefish sequence. A distinct python script, compared to that used for the main ohnolog pair analysis, was employed to classify the sturgeon-paddlefish subtree into either PreSpec-type or PostSpec-type . To better understand the distribution of PreSpec-type and PostSpec-type trees observed we considered whether the paddlefish genes in PostSpec-type trees  may in fact result from collapsing of two ohnologs into a single assembly region rather than being single-copy. To test this, we mapped paddlefish genome DNA-sequencing reads  the two sturgeon-paddlefish ortholog pairs from each PreSpec gene tree, and (ii) the Single-Copy Orthogroups present in all species as inferred in our OrthoFinder analysis. This latter set of Single-Copy Orthogroups could potentially incorporate some hidden ohnologs if there are cases of differential PreSpec ohnolog loss between sturgeon and paddlefish, and so is more prone to bias, but still a useful comparison. In each case the corresponding coding sequence to the amino acid sequences used in gene tree analyses were applied, and pairs where either sequence was flagged with a warning by wgd were removed and wgd reran. Pairs with Ks values ≥ 0.3 were not included in the final data for visualisation. To supplement analyses of ortholog pair peak Ks differences between paddlefish and sturgeon, sturgeon-paddlefish ortholog branch lengths were compared to assess for differences in amino acid evolutionary rate in substitutions per site in each species. For this analysis, we ensured that all gene trees were rooted in accordance with wider jawed vertebrate phylogeny and that sturgeon and paddlefish sequences formed a monophyletic grouping (as per Ohnolog pair datasets methods subsection above). We then extracted branch lengths for sturgeon and paddlefish from each of the two PreSpec sturgeon-paddlefish orthologs, as well as from Single-Copy Orthogroups  using a custom ETE toolkit python script. The sturgeon-paddlefish clade monophyly check and focus on PreSpec orthologs and (to a lesser extent) Single-Copy Orthogroups should ensure that divergence of the sturgeon and paddlefish sequence pair reflects ortholog divergence since speciation. Thus, variation in these branch lengths between species specifically captures substitution rate variation. A caveat to this exists for Single-Copy Orthogroups, where differential ohnolog loss producing hidden ohnologs could mislead analyses of this dataset. We compared the sturgeon and paddlefish branch lengths using the paired-sample Wilcox-test in R (version 4.1.2 [2021-11-01])91. Extreme outlier ortholog pairs with branch length values ≥ 0.15 were not included in the final data for visualisation or statistical comparison.The ksd command from the wgd tool100) of the sturgeon and paddlefish genomes. All members of an ohnolog pair were required to be present on the largest 60 chromosomes to be included. For plotting of both species in a single circos plot, PreSpec ohnolog pairs were split into separate ohnologs to be plotted as orthologs between species, while PostSpec ohnolog pairs were plotted as ohnologs within species as per the individual species plots.The genomic coordinates of each gene in an ohnolog pair were used to anchor links between ohnologs on circos plots . To simplify preparation for phylogenomic analysis and reduce computation time of dating analyses, we then filtered for gene families that were otherwise single copy, resulting in a set of 81 gene families. We generated five distinct datasets by randomly assigning ohnologs from a pair as the A or B copy prior to concatenation of all 81 existing gene family multiple sequence alignments. This avoids bias from a single arbitrary concatenation, while also permitting assessment of how robust results are to variations in ohnolog concatenations101 (-nogaps) and BMGE102 (-m BLOSUM62) to trim gap-rich and saturated sites, after which 42,126 amino acid alignment sites remained in each dataset.Upon concatenation each super-matrix was then filtered using trimAl63 (version 4.1c), specifying the site-heterogeneous CAT-GTR + G465 substitution model along with an autocorrelated lognormal relaxed clock model64 (which fits and performs well in jawed vertebrate phylogenomics85), and a birth-death prior with soft bounds104 on fossil calibrations. Fossil calibrations priors ,)),,),,)))));”) was specified based on accepted jawed vertebrate phylogeny85 and our inference of a shared WGD, and the Chondrichthyes representatives, ghost shark and whale shark, were set as the outgroup. We verified this topology for each of our five datasets by performing a basic concatenated phylogenomic analysis in IQ-tree86 under the JTT + G4 model87 with 1000 UFBoot bootstrap replicates48.Phylogenomic divergence dating was then performed  in PhylobayesEach Phylobayes Markov chain Monte Carlo analysis was sampled for at least 10,000 cycles, with the first 5000 discarded as burn-in before calculation of inferred divergence dates and 95% credibility intervals. Runs under the prior were performed using the same settings, except for swapping to a site-homogeneous Poisson substitution model for computational efficiency since the prior over divergence times is independent of substitution model priors, to verify that the prior on the sturgeon-paddlefish WGD lower bound timing were sufficiently diffuse as to be uninformative.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Aortic valve replacement (AVR) is a frequently performed procedure for treating aortic valvulopathy. AVR involves replacing the damaged aortic valve with either a mechanical or a bioprosthetic valve. While many factors are involved when selecting between the two options, age and patient preference are the deciding factors at this point. Mechanical valves demonstrate long-standing durability that overlaps with the accompanied bleeding risk due to lifetime anticoagulant therapy, making them a more favorable choice for younger patients. Bioprosthetic valves are preferred for older patients as they show a reduced risk of thrombogenicity. However, bioprosthetic valves have a higher incidence of structural valve degeneration (SVD) than mechanical valves. Our case report focuses on a 76-year-old patient who had undergone an AVR with a bioprosthetic valve at the age of 33, which has still not demonstrated any valve deterioration. As the longest known case of bioprosthetic durability, this patient provides useful data for designing bioprosthetic valves more resistant to structural degeneration and thereby better suited to younger patients or those at higher risk of bleeding. Aortic valve replacement (AVR) is one of the most common cardiac procedures performed and is used to treat mainly aortic stenosis but can also be used for aortic regurgitation . AVR is Age is a vital factor to take into consideration when choosing between mechanical and bioprosthetic valves for AVR . MechaniHowever, mechanical valves are thrombogenic and therefore require long-term treatment with anticoagulants, specifically warfarin, to prevent clots, leaving patients at risk of bleeding and enforcing diet and activity restraints . To avoiAchieving this goal requires up to 20 years’ worth of data on the cases of long-term optimal hemodynamic function with no structural deterioration, which is currently unavailable . AdditioA 76-year-old male patient presented to the cardiologist’s clinic for a three-month follow-up. He was evaluated by the cardiologist and was deemed clinically stable. The patient has a medical history of hypertension, anemia, arthritis, hyperlipidemia, gout, and valvular heart disease. His current medications include alendronate 70 mg weekly, calcium citrate 1200 mg daily, folic acid 1 mg daily, lisinopril 10 mg daily, atorvastatin 20 mg daily, tamsulosin 0.4 mg daily, vitamin D 50000 IU weekly, and niacin extended release (ER) 1000 mg daily. He is also a former smoker, having quit smoking at the age of 48. As shown in Figure The results from a transthoracic echocardiogram done on May 1, 2023, showed moderate enlargement of the left ventricle with decreased systolic function, global hypokinesis with an ejection fraction of about 35%-40%, mild to moderate left ventricular hypertrophy (LVH), E/A reversal (diastolic dysfunction), asymmetric septal hypertrophy, mild left atrial enlargement, mild mitral regurgitation (MR), mild tricuspid regurgitation (TR), and the prosthetic aortic valve. The aortic valve appears to be functioning adequately and appropriately. An EKG from October 2022 showed sinus rhythm with left axis deviation, poor R wave progression, and intraventricular conduction delay  is a recent advancement that allows for the removal of the damaged valve while eliminating the need for sutures, as well as minimizing aortic cross-clamp and cardiopulmonary bypass (CPB) time, reducing the potential risk of cardiovascular morbidity and mortality . While tMultiple studies indicate that patients younger than 65 years of age experience increased rates of structural valve degeneration after AVR with a bioprosthetic valve, regardless of whether a stented or stentless valve was used ,9. IncreThe incidence of SVD restricts the longevity of bioprosthetic valves. One study found that 21.5% of the patients with subclinical SVD either advanced to clinical SVD or underwent additional aortic valve surgery within a 26-month period. These patients tended to be younger and more prone to hypertension, indicating the influence of age on the rate of SVD . Valve lTransthoracic echocardiography (TTE) is the standard method to assess prosthetic valve function and diagnose SVD. Currently, both the European Society of Cardiology and the American Heart Association/American College of Cardiology guidelines recommend establishing a baseline assessment within 6-12 weeks and long-term monitoring via annual TTE starting five or 10 years post-procedure, respectively. However, since follow-ups by repeated TTE are undertaken only if the patient manifests symptoms suggesting valve dysfunction, early subclinical changes in valve performance may be missed, and Rodriguez-Gabella et al. recommend that at least one TTE should be done between the one- and five-year follow-ups followed by annual examinations thereafter . AdditioDifferent bioprosthetic valves have been designed to achieve maximum durability in patients. The implementation of the stentless Freestyle bioprosthetic valve, utilized in aortic root replacements, was regarded as successful as it ensured long-standing clinical stability and hemodynamic performance for patients older than 60 . MortaliThis case report focuses on a 76-year-old patient who had an AVR with an SJM bioprosthetic valve in 1980, indicating that the valve has withstood a duration of more than 42 years without known structural valve degeneration. Upon examination, it was discernible that the patient retained clinical and hemodynamic stability and was free of symptoms associated with valvular dysfunction, allowing for daily activities without restraint. Although a majority of patients younger than 65 experienced deterioration of their bioprosthetic valves within a decade, this patient’s valve has not undergone SVD despite the patient being 33 years old at the time of the AVR [The evidence of cases such as these can establish the potential for improved durability of bioprosthetic valves and expand their use in younger patient populations. The patient that is the focus of this case study is a prime example of bioprosthetic longevity post-AVR and may be the longest survival case known. This adds to the scientific literature by providing data that can identify key characteristics indicative of reducing the occurrence of SVD, resulting in prospective improvements in bioprosthetic valve design. Additionally, this case can guide clinicians to consider alternatives to mechanical valves for younger patients, avoiding long-term anticoagulant therapy and reducing the risk of bleeding. Furthermore, this case study demonstrates that bioprosthetic valves used in AVR may be more durable in younger patients than was previously acknowledged, presenting the possibility of the expansion of valve options.


This case contemplates the unusual presentation, challenging diagnostic workup and conservative therapeutic process of a patient with Actinomyces empyema complicated along the way due to drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome. The patient was a 40-year-old male, who presented with pleurodynia and fever. Laboratory exams showed elevated inflammatory markers and imaging revealed two biconvex fluid pockets located in the right lower lobe, from which the fluid was positive for Actinomyces meyeri. The initial conservative process with intravenous antibiotics and successful drainage with intrapleural fibrinolysis improved our patient. However, after a few days, the patient’s fevers relapsed, and as regress of the empyema was discussed as a complication, he developed a maculopapular symmetrical rash of the trunk and legs accompanied by enlarged lymph nodes, eosinophilia, thrombocytopenia, and atypical lymphocytes. The diagnosis of DRESS syndrome due to antibiotic therapy for actinomyces empyema was established and a balance between bactericidal and immunosuppression medication had to be found. Fortunately, the patient withstood prolonged antibiotic therapy and got fully treated without any relapses. Pulmonary actinomycosis frequently develops in middle-aged males, especially those with a history of alcohol abuse and structural lung diseases. The clinical appearance is nonspecific and includes fever and chest pain . DiagnosA 40-year-old shepherd  was referred to the emergency department because of right hemithorax pleurodynia and fever. At pleurodynia onset one month prior, the patient was prescribed analgesics and muscle relaxants for symptomatic relief. However, five days before his visit to the emergency department the patient became febrile and completed a course of moxifloxacin. No remarkable medical history was obtained except for daily consumption of alcohol and a tooth abscess that resolved spontaneously two months ago. The patient was admitted to the Department of Respiratory Medicine to be diagnosed and treated.On admission, the patient was febrile (temperature 39.4oC). Peripheral lymph nodes were normal. During lung auscultation, right middle-lung-area coarse crackles were recognized while the right lower lung field was silent, devoid of any breath sounds with dullness upon percussion. No pathological heart sounds were detected. Oropharynx inspection revealed gingivitis and dental caries. No other abnormalities were found. The patient had hypoxemic respiratory failure  according to arterial blood gas analysis. Remarkable laboratory findings involved leukocytosis , anemia , reactive thrombocytosis (780 x 103 platelets/μL), elevated c-reactive protein  and increased erythrocyte sedimentation rate (104 mm/hr).Chest radiography revealed a blunting of the right costodiagraphmatic recess and a triangular opacification in the right middle lung field by the visceral pleura Figure . Chest cAs for further diagnostic studies, transthoracic echocardiography did not show evidence of infective endocarditis, blood and sputum cultures were negative, however microbiological and molecular studies of the pleural fluid were positive for Actinomyces meyeri. The chest tube within pocket 1 was removed following successful drainage after three courses of intrapleural fibrinolysis which resulted in defervescence & improved gas exchange. After three weeks of intravenous piperacillin-tazobactam for extended anaerobic coverage beyond actinomyces, however, the fever relapsed. A neck/chest computed tomography was negative for cervicofacial or mediastinal actinomycosis and showed signs of empyema resolution Figure .The patient developed a maculopapular symmetrical rash of the trunk and legs Figure , 4B, 4C After the diagnosis, piperacillin/tazobactam was immediately discontinued. Intravenous immunoglobulin and pulses of intravenous prednisolone were initiated due to concerns that sinus bradycardia could be a herald of eosinophilic myocarditis. After five days of intravenous prednisolone 125mg) pulses and as soon as the skin rash showed clinical improvement and sinus bradycardia reverted to sinus rhythm, intravenous doxycycline was carefully initiated as a substitute for b-lactam therapy. Corticosteroids were quickly tapered off after carefully considering the risk of progression of actinomycosis and DRESS relapse. The patient received a total of four weeks of intravenous antibiotic therapy under close in-hospital supervision and was then discharged and prescribed oral doxycycline  effective early drainage through a chest tube; ii) no evidence of extensively disseminated disease; iii) absence of hemoptysis or bronchopleural fistulae; iv) no immediate need for decortication and v) adequate response to therapy. When the fever relapsed after three weeks of treatment, the first thought was Actinomycosis deterioration. Only after the improved CTs, the peripheral blood eosinophilia, and the rash appearance, the DRESS syndrome was suspected and then diagnosed.Actinomycosis is a rare, chronic bacterial infection that induces suppurative, granulomatous inflammation. Involvement of the lung is evident in 15% of total cases and is thought to occur secondary to aspiration of oropharyngeal or gastrointestinal secretions . ActinomDefinitive diagnosis is elusive given the non-specific clinical/radiological findings and the challenging isolation of the bacteria from readily available clinical samples such as sputum and pleural fluid. The presence of sulfur granules in purulent material is a pathological hallmark of actinomycosis however they are also found in other conditions such as nocardiosis, coccidioidomycosis, and aspergillosis . Upon hiSurgery is useful in controlling severe complications such as recurrent hemoptysis, bronchopleural fistulae, or restrictive lung disease following empyema. Invasive procedures may even be required when cancer cannot be ruled out or when antibiotic treatment alone fails to provide sufficient benefit. When promptly diagnosed, a significant number of patients respond favorably to treatment with high-dose intravenous penicillin for two to six weeks and then a prolonged regimen of penicillin V or amoxicillin for six to 12 months. The duration and administration route is decided on a case-by-case basis according to disease severity and clinical course ,11. MoreDespite the well-documented safety of long-term b-lactam use in pulmonary actinomycosis patients, rare and even life-threatening side effects may develop. DRESS syndrome describes a hypersensitivity reaction to certain medications usually two to six weeks after exposure. The condition primarily involves rash, lymphadenopathy, fever, and characteristic hematologic abnormalities . Elevated transaminases or hepatomegaly are also very common in light of the liver being the main site of internal organ involvement . AlthougWe believe this work will help raise further awareness about rare infections such as actinomyces empyema complicated with severe allergic reactions such as DRESS syndrome and will be of particular interest to chest physicians as they are more likely to encounter such cases. High clinical suspicion is imperative in the setting of actinomycosis as timely identification favors effective resolution with long-term antibiotic therapy without the need for surgical intervention. During or after the antibiotic therapy, any findings of skin rash, eosinophilia, elevated transaminases, and relapse of fever should raise suspicion for drug reaction with eosinophilia and systemic symptoms syndrome. In that case, immediate discontinuation of the offending agent and systemic corticosteroids are the main options.


Breast-conserving surgery is aimed at removing all cancerous cells while minimizing the loss of healthy tissue. To ensure a balance between complete resection of cancer and preservation of healthy tissue, it is necessary to assess themargins of the removed specimen during the operation. Deep ultraviolet (DUV) fluorescence scanning microscopy provides rapid whole-surface imaging (WSI) of resected tissues with significant contrast between malignant and normal/benign tissue. Intra-operative margin assessment with DUV images would benefit from an automated breast cancer classification method.Deep learning has shown promising results in breast cancer classification, but the limited DUV image dataset presents the challenge of overfitting to train a robust network. To overcome this challenge, the DUV-WSI images are split into small patches, and features are extracted using a pre-trained convolutional neural network—afterward, a gradient-boosting tree trains on these features for patch-level classification. An ensemble learning approach merges patch-level classification results and regional importance to determine the margin status. An explainable artificial intelligence method calculates the regional importance values.The proposed method’s ability to determine the DUV WSI was high with 95% accuracy. The 100% sensitivity shows that the method can detect malignant cases efficiently. The method could also accurately localize areas that contain malignant or normal/benign tissue.The proposed method outperforms the standard deep learning classification methods on the DUV breast surgical samples. The results suggest that it can be used to improve classification performance and identify cancerous regions more effectively. ThesThe evaluation of the intra-operative margin is currently performed by radiographic examination and confirmed by post-operative histological analysis of hematoxylin and eosin (H&E) images. As a preferable alternative, having a microscopic imaging system for margin assessment during surgery is beneficial. Real-time whole-surface imaging (WSI) of resected tissues during BCS can be provided by deep ultraviolet fluorescence scanning microscopy (DUV-FSM) , 5. DUV-In recent years, the medical domain has explored deep-learning methods for disease detection. Image classification algorithms can aid in detecting and localizing disease regions of interest (ROI). Breast cancer classification and margin assessment are major research areas that have shown promising results , 7. For The researchers mentioned above trained and tested the network on a publicly available H&E image dataset. At the moment, there is difficulty in obtaining a large training set of DUV images due to its relative novelty and practical application on a small group of subjects. Well-trained models may generalize poorly on a limited DUV dataset. To prevent the problem of overfitting, the DUV WSI images are divided into small, non-overlapping patches. In this way, the network will be able to learn better generalizations. This patch methodology boosts the training data at the patch level and helps localize cancer regions in DUV WSI for margin evaluation. A transfer learning approach is utilized to extract convolutional features from DUV patches with a pre-trainedconvolutional neural network and input them into decision-tree-based classifiers for DUV patch-level classification. As an ensemble approach, the patch-level classification results are merged with regional importance map calculations using Gradient-weighted Class Activation Mapping++ (Grad-CAM++)  into a s22.1A DUV WSI is divided into multiple DUV patches. The where Since each DUV WSI has different dimensions, the images are resized to the closest dimensions divisible into non-overlapping patches The minimal alterations to the dimensions should not affect the quality of the morphological characteristics, such as cell density and infiltration, because the DUV WSI images are very large. Afterward, a 2D grid system is implemented based on each sample’s FOV ExtractPatches, and GetPercentage.The patch images are extracted from each non-overlapping patch ExtractPatches is used to extract foreground patches for each DUV WSIAlgorithm 1  DUV WSI sample  2D grid system on each sample’s FOV Non-overlapping patch with  pixels valid patch image number of DUV WSI samplesfor  do Load in sample  for  in  do  if GetPercentage  then   Save  as valid patch  for   end if  end forend forGetPercentage is used to determine the foreground percentage of a patchAlgorithm 2 ImageSize  pixelsPixelCounter for  do for  do  if Image  BackgroundThreshold then   PixelCounter  PixelCounter   end if end forend for return PixelCounter  ImageSizeBackgroundThreshold 2.2Convolutional neural networks are widely used for image classification problems and have the potential to diagnose diseases. In architectures like ResNet, the ‘vanishing gradient’ problem exists. The architecture of a standard ResNet50 is visualized in For this issue, a transfer learning approach determines whether each patch is malignant or normal/benign for all  network  on the I network . The XGb2.3Grad-CAM++  is an exAs shown in this equation, the where The novelty of DenseNet is that it removes repeated feature maps after the concatenation of the feature maps, allowing for fewer parameters. By concatenating these feature maps from different feature maps, it increases the variation in the inputs of the following layers. The bottleneck and compression layers of the DenseNet architecture are effective against overfitting due to fewer parameters needed. The bottleneck layers help reduce the number of inputs from previous The following equations describe Grad-CAM++’s process of extracting weight maps from feature maps. The final classification score is defined as The gradient of the classification score where With Grad-CAM implementations, visualizations are limited if there are multiple instances of a class in the input image To fix this issue, a weighted average of the pixel-wise gradients can be taken instead, where Equation 3 is restructured as:where The gradient weights Without a threshold need, the To isolate The Grad-CAM++ weights are calculated by substituting Equation 7 in Equation 4:The regional importance map This highlights the most significant features in the final classification with a positive correlation with pixel intensity and classification score with the application of the 2.4Given the patch-level classification labels First, the regional importance where The weight is defined as This weighting scheme ignores the patches with low importance, either malignant or normal/benign, in the fused decision for the DUV WSI.Next, the patch-level classification label Now, the total amount of malignant patches Finally, the WSI-level classification label where it maps the positive  and negative (benign) value to 3Automated DUV WSI classification is used to determine if a sample is malignant or normal/benign tissue. The proposed method was assessed with 5-fold cross-validation. The DUV WSI data was split while managing balanced class labels and specific patient samples to stay only in training or testing data splits. There was no fine-tuning of the pre-trained ResNet50 with ImageNet weights for the Transfer Learning part. The XGBoost classifier’s hyperparameters were default settings and did not involve any tinkering. There was no hyperparameter tuning for the pre-trained DenseNet169 model with ImageNet Weights for the Regional Importance Calculation. The proposed approach was compared with a standard ResNet50 model and Patch Classification with Majority Voting. The ResNet50  trained 3.1The breast cancer dataset consists of DUV images from 60 samples . This DUV dataset was collected from the Medical College of Wisconsin (MCW) tissue bank  and normal/benign adipose (green) tissue. These DUV images show that the proposed fusion method with regional importance maps can localize ROI for a WSI-level decision with confident margin prediction accuracy. The results demonstrate that even with little training data, this deep learning approach with a patching strategy can capture pathological traits like high cell density and infiltration.3.3The accuracy, sensitivity, specificity, and AUC score of malignant/benign binary classification on DUV-WSI images are measured for quantitative analysis. The classification performance of 5-fold cross-validation across 60 DUV-WSI images is shown in where Compared to standard ResNet50, the proposed method significantly improves classification performance with a 13.3% increase in accuracy. The proposed method is reliable, while the standard ResNet50 with DUV WSI has overfitting issues. The proposed method outperforms ResNet50 in sensitivity, specificity, and AUC scores by 8.3%, 20.8%, and 17.0%, respectively. 3.4The three normal/benign DUV WSI images that were misclassified as malignant are shown in 4This part discusses the results of the proposed breast cancer classification method for DUV image margin assessment, which combines patch-level classification using a transfer learning approach with regional importance maps generated by the Grad-CAM++ algorithm. The discussion involves the effectiveness of the method, its limitations, and potential future research directions to improve intra-operative margin assessment during breast-conserving surgery.4.1This study used a pre-trained ResNet50 model with ImageNet weights to extract convolutional features from DUV WSI patches, which were then used to train an XGBoost classifier for patch-level classification. The regional importance maps were generated using the Grad-CAM++ algorithm on a pre-trained DenseNet169 model with ImageNet weights to localize crucial areas in the WSI image for margin assessment. The overall WSI label was determined by fusing the patch-level classification results with the regional importance map, which enhanced classification accuracy at the WSI level. The proposed method achieved an accuracy of 95% in determining DUV WSI and displayed 100% sensitivity in detecting malignant cases. Additionally, the approach could accurately localize malignant and normal/benign tissue areas, outperforming standard deep-learning classification methods on DUV breast surgical samples.4.2in situ carcinoma, and invasive carcinoma. For the experiment, 320 samples were assigned for training/validation, and 80 samples were assigned for testing with balanced classes. To address the issue of limited data and large image sizes in the BACH dataset, patches of size 512 x 512 pixels were extracted with a 50% overlap between patches, resulting in a final amount of 14,000 patches. The training/validation set was augmented by applying data augmentation through varying degrees of rotation and flipping the extracted patches. The remaining 80 samples were used as a test set to evaluate the accuracy of classification methods. The approach achieved a whole-image classification accuracy of 97.50%, and a majority voting process was employed to classify breast histology images into four tissue sub-types: normal, benign, in situ carcinoma, and invasive carcinoma, based on patch-wise class labels estimated using the network. This ResNet50-based method demonstrated the powerful classification capacity of transfer learning approaches for automatically classifying breast cancer histology images, especially when dealing with limited training data.A previous study utilized a transfer learning-based approach with ResNet50 to classify H&E-stained histological breast cancer images from a different dataset . The metThe proposed ensemble learning-based approach showcases its robustness, even with a limited dataset, offering advantages comparable to the ResNet50-based method. In contrast, the proposed method focuses on margin assessment during breast cancer surgery using the DUV WSI dataset. The method uses a pre-trained ResNet50 model with ImageNet weights to extract convolutional features from DUV WSI patches. These extracted features are then used to train and test an XGBoost classifier for patch-level classification, providing a more robust and accurate representation of the input data. Combining the pre-trained ResNet50 model and the XGBoost classifier effectively utilizes the limited dataset available while minimizing overfitting, a critical aspect when dealing with a small dataset like the one used in the study.The proposed ensemble learning method offers several advantages compared to the ResNet50-based approach. One key advantage is incorporating regional importance maps generated using the Grad-CAM++ algorithm on a pre-trained DenseNet169 model with ImageNet weights. These regional importance maps help focus on areas with the highest level of detail and semantic information within the DUV WSI images. By capturing the spatial distribution of the most discriminative features, the algorithm highlights the most critical regions in the image responsible for the classification decision. This emphasis on semantically significant areas substantially contributes to breast cancer margin assessment, enabling the method to hone in on the most relevant image regions that could contain malignant or benign tissue.in situ carcinoma, and invasive carcinoma. While this approach provides valuable information regarding the tissue types present, it may not directly apply to margin assessment in the same way as the proposed method, as it requires additional interpretation to decide the margin status.The proposed method aims to facilitate more accurate and efficient decisions during breast cancer surgery by concentrating on the critical aspects of margin assessment. This additional step offers a more streamlined decision-making process tailored for margin assessment during breast cancer surgery. In addition to patch-level classification, the proposed approach assigns a malignant or normal/benign label to each WSI. By focusing on this binary classification, the proposed method simplifies the task for surgeons, making it easier to determine whether the margins are clear or require further resection. On the other hand, the ResNet50-based method classifies images into four tissue types: normal, benign, Although a direct comparison between the two approaches may not be fair due to their fundamental differences, the proposed method highlights the potential advantages of deep learning techniques in breast cancer margin assessment using DUV images. By utilizing transfer learning, a robust gradient-boosting algorithm, and regional importance maps, the proposed approach demonstrates the effectiveness of deep learning in accurately identifying cancerous regions in DUV WSI images. This method paves the way for further research and development in this area, potentially contributing to improved surgical outcomes and reduced breast cancer re-excision rates after BCS.4.3The dataset used in this study was relatively small, consisting of 60 images out of 66 available images. This limitation may restrict the generalizability of this study’s findings, as the model’s performance on more extensive or diverse datasets still needs to be investigated. Training a robust network without overfitting on such a small dataset can also be challenging, requiring careful selection of training parameters and model architecture.Another challenge encountered in this study is the presence of mixed tissue types within many tissue samples. The label assigned to each patch may only represent a portion of the area within the patch, potentially complicating the classification process. Determining the optimal patch size remains an open question; better classification results could be achieved by identifying and using an optimal patch size that maximizes the model’s discriminatory power.This dataset mostly contained samples that were either predominantly benign/normal or malignant, with six samples exhibiting a complex mixture of benign/normal and malignant tissues being disregarded for the sake of model training. While this approach trains the model on clean and pure samples, it may limit the generalizability of the proposed method to samples with a greater complexity of benign/normal and malignant tissues. Consequently, further investigation of the model’s performance on more complex and diverse samples is essential to validate its efficacy and robustness in real-world scenarios.4.4Future research can validate findings using larger and diverse datasets, optimizing patch size for classification to enhance performance, and exploring alternative evaluation metrics for more comprehensive comparisons. Assessing the clinical impact of the proposed method can guide surgical decision-making, potentially reducing the need for re-excision. Integrating the proposed method into surgical navigation systems can improve precision and minimize damage to healthy tissues. Investigating the method’s performance on other medical imaging modalities can enhance its versatility and applicability to different diagnosis techniques, contributing to better patient outcomes.5Accurate margin assessment during breast cancer surgery is crucial for reducing breast cancer re-excision rates following breast-conserving surgery (BCS). This study addressed the challenge of locating cancerous regions in DUV whole-slide images (WSI) by proposing an automated method leveraging deep learning techniques’ power. This methodology combines patch-level classification using transfer learning with regional importance maps generated through Grad-CAM++. Focusing on highly significant regions within the images assigns a malignant or normal/benign label to each WSI with increased confidence. This approach enhances the robustness of breast cancer classification in DUV WSI images, which is essential for accurate intra-operative margin assessment. The proposed method was evaluated on a dataset of 60 authentic DUV WSI images, and an impressive classification accuracy of 95.0% was achieved. This result indicates the potential of the proposed method for real-time assessment of margin status during breast cancer surgery and potentially other types of cancer surgeries. However, it is essential to acknowledge that the study has limitations, such as the relatively small dataset size and potential generalizability challenges for more complex tissue samples. Future work could focus on expanding the dataset, optimizing patch size, and investigating alternative evaluation metrics to validate further and enhance the proposed method. The approach used in this study could impact clinical decision-making significantly. It can provide real-time guidance to surgeons during operations, ensuring they make more informed decisions about resection margins. It can also help develop personalized treatment plans. Overall, this study demonstrates the effectiveness of deep learning techniques in breast cancer margin assessment using DUV WSI images. Furthermore, this method can be integrated into surgical navigation systems to visualize cancerous regions and their boundaries in real time, improving the precision of surgical interventions and minimizing damage to healthy tissues. The potential clinical applications and the possibility for further refinement of this method offer a promising direction for future research and development in medical imaging.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Conceptualization, TY, BY, and DY; data curation, TL, MP, JJ, and TS; funding acquisition, TY, BY, and DY; investigation, TT, and DY; methodology, TT, and DY; software, TT; writing—original draft, TT; writing—review and editing, BY, and DY. All authors have read and agreed to the published version of the manuscript.


We present a 52-year-old male patient with cardiogenic shock who was placed on veno-arterial extracorporeal membrane oxygenation (ECMO) as a bridge to an orthotopic heart transplant. While on ECMO, the patient developed an acute intracranial bleed confirmed on computerized tomography (CT). However, his clinical status deteriorated and he was unstable for transport to evaluate for worsening hemorrhage. Instead, optic nerve sheath (ONS) ultrasonography was utilized to confirm increased intracranial pressure, which guided the goals of care until he stabilized enough to transport for advanced imaging. Repeat CT confirmed the worsening of his cerebellar bleed with obstructing hydrocephalus and brainstem compression. This case demonstrates how ONS ultrasound can be utilized in a cardiothoracic intensive care unit to evaluate sedated patients for new or worsening intracranial hemorrhage. In ECMO patients, who are often unstable with the risks of transportation for CT outweighing potential benefits, ONS ultrasonography can provide the care team with meaningful data on a patient’s neurologic status. Extracorporeal membrane oxygenation (ECMO) is a vital tool for severe respiratory and circulatory failure. Unfortunately, veno-arterial (VA) and veno-veno (VV) ECMO are not without significant risk. Patients on ECMO are often coagulopathic. Combined with high-dose anticoagulation -most often unfractionated heparin - these patients have an increased intracranial hemorrhage (ICH) risk per the Extracorporeal Life Support Organization (ELSO) Registry. In the 3% of ECMO patients that develop an ICH, the mortality risk increases from 56% to 86% and 36% to 73% for VA and VV ECMO, respectively -2. CompaAs with any significant bleed, ICH is a devastating complication, associated with substantial mortality and disability. Diagnosing ICH early allows for careful decision-making of whether to pursue treatment or withhold potentially scarce treatment resources. However, effective screening for ICH is limited in ECMO patients by sedation and the inability to perform a neuro exam. Despite a non-contract computerized tomography (CT) scan of the head being the gold standard diagnostic tool for ICH, transporting ECMO patients for imaging is not without risk. Over 30% of transports are affected by adverse events, such as significant bleeds, unstable hemodynamics, or equipment malfunctions  .Another option would be optic nerve sheath (ONS) ultrasonography, which has been validated for use in detecting elevated intracranial pressure (ICP). The ONS is particularly sensitive to increases in ICP, with an increased ONS diameter having a pooled sensitivity of 0.9 and specificity of 0.85 for elevated ICP [A 52-year-old male patient with a pertinent past medical history of myocarditis secondary to acute coronavirus disease 2019 (COVID-19) infection and heart failure with a reduced ejection fraction of 20-25% presented in cardiogenic shock. His shock state was suspected as being secondary to his non-ischemic cardiomyopathy and the patient was placed on VA ECMO via femoral artery/femoral vein approach while undergoing evaluation for an orthotopic heart transplant. The patient’s course was complicated by arrhythmias, acute renal failure requiring continuous renal replacement therapy, intracardiac thrombus formation, and bloody nasogastric output from a presumed gastrointestinal bleed. To further assess potential gastrointestinal bleeding, the patient was sent for a CT Abdomen. Up to this, point he had consistently demonstrated a normal neuro exam when sedation was held; however, a CT Head was also ordered given our intermittent inability to discontinue sedation without soliciting agitation. Sedation was maintained with propofol to limit agitation. The initial CT Head demonstrated an acute intraparenchymal hemorrhage in the left cerebellar hemisphere, measuring 3.5 cm x 1.5 cm, with partial effacement of the fourth ventricle  ultrasound probe. Tegaderm was applied over closed eyes with adequate ultrasound gel layered above the dressings. The ultrasound probe was then fanned through transverse planes of the eyes, minimizing pressure to the eyes, until the mid-axial planes were identified. With the optic nerves visualized in the mid-axial planes, 3 mm was measured posterior to each globe, followed by measuring the distance across the hypoechoic nerve sheaths Figure . The pro MHz ultrAs the optic nerve is continuous with the central nervous system, its meningeal sheath is an extension of the dura mater and will dilate as increasing ICP causes an influx of cerebrospinal fluid. In a meta-analysis performed by Robba et al., the accuracy of an ONS diameter of ≥ 0.6 cm for identifying ICPs > 20 mmHg ranged from 0.811-0.954, with an area under the receiver-operating characteristic curve of 0.938 . MeasureCurrently, there is no standardized method of screening ECMO patients for neurologic injury. While the National Institute of Health Stroke Scale has been proposed as a standardized means of detecting focal deficits, this method is not always applicable in the case of insidious onset of injury or the inability to wean sedation . CT remaWhile its accuracy in detecting elevated ICP has been validated in neurosurgical ICUs against invasive intracranial pressure monitors such as ventriculostomy and intraparenchymal microtransducers -10, sonoThis case highlights the potential utility of ONS ultrasound in detecting new or worsening changes in intracranial pressures in adult patients on ECMO. With these patients often too unstable for transportation to obtain CT imaging, an alternative, safer tool for guiding care decisions may be optic nerve ultrasonography. While heavily explored in neurological ICUs, to date there have been no published studies analyzing how this methodology could be applied to improve patient care and outcomes in adult cardiothoracic ICUs. Therefore, further prospective data should be collected to evaluate the benefit of including optic nerve sheath ultrasounds as part of the standard assessment for patients on ECMO.


TBCD) gene, involved in the assembly and disassembly of the α/β-tubulin heterodimers, have been reported in early-onset progressive neurodevelopment regression, with epilepsy and mental retardation. We describe a rare homozygous variant in TBCD, namely c.881G>A/p.Arg294Gln, in a young woman with a phenotype dominated by distal motorneuronopathy and mild mental retardation, with neuroimaging evidence of corpus callosum hypoplasia. The peculiar phenotype is discussed in light of the molecular interpretation, enriching the literature data on tubulinopathies generated from TBCD mutations.Mutations in the tubulin-specific chaperon D ( TBCD gene is located in the chromosome 17q25.3 and codes for a protein of 1193 amino acids termed tubulin folding cofactor D. The TBCD protein is mainly composed of armadillo/HEAT motifs or multiple α-helices running in antiparallel directions linked by short loops and a TFCD-C C terminal. It contains three HEAT repeats  only recently, following a single case report [Mutations in the genes encoding tubulins and microtubule-associated proteins cause different neurodevelopmental and early-onset neurodegenerative disorders ,9,10. The report .TBCD gene in a woman with a phenotype resembling distal motorneuronopathy. We discuss the putative role of this gene in our case in light of the molecular and previous literature data.Here, we report a rare homozygotic mutation in the A 22-year-old woman presented with early fatigue and low exercise tolerance since childhood. She was born from a full-term eutocic birth. On personal history, she reported a slight intellectual disability with an Intelligence Quotient at age 17 measured by WAIS-IV of 61, mainly driven by defective linguistic (63) and comprehension (63) skills, childhood social anxiety disorder with selective mutism and behavioral disorder, and pavor nocturnus until age 8. Her history was negative for epilepsy, and her EEG record was normal. The motor and developmental stages of development were normally achieved. Upon orthopedic assessment, initially the symptoms were ascribed to the feet deformity (flatfeet), leading to bilateral corrective surgery when she was 13. Given the persistence of symptoms after surgery and physical rehabilitation, a neurological examination at 17 years old was performed, showing mild distal weakness, without visible fasciculations, associated with hypotrophy in the intrinsic muscles of the hand and posterior–anterior distal leg muscle lodges A,B. The Neurophysiological study showed sporadic fibrillations potentials and positive sharp waves in the left I dorsal interosseous and right anterior tibial. The motor unit potentials were globally of large amplitude and long duration, with sporadic polyphasic potentials. Decreased motor unit recruitment was revealed, thus revealing a reduced number of motor units. The motor evoked potentials revealed a prolongation in the peripheral motor conduction time.The cervical Magnetic Resonance Imaging (MRI) was normal, whereas a brain MRI repeated at age 21 revealed a thin corpus callosum , which wSlightly elevated creatine kinase (CK) values (217 UI/mL) were reported in the serum. An extensive autoimmune screening was uninformative. In looking for a systemic involvement, the abdomen and thoracic ultrasound and eye examination revealed no abnormalities. The transthoracic heart ultrasound showed a slight left ventricular wall thickening without any dilatation or alterations in segmental kinetics. The ascending aorta and all the valves were normal.The disease slowly progressed over the years, and the patient mainly complains of fatigue in several daily actions, including climbing the stairs and walking for long distances, without shortness of breath. She did not report any difficulties with the fine movements with her hands. The family history  revealedSPAST gene and the direct sequencing of the SMN  gene did not show any deletions or mutations.Informed consent for genetic analysis was obtained from the patient. Genomic DNA was extracted from the peripheral lymphocytes obtained from the patient as standard procedures . The MLPTBCD. The variant was confirmed by Sanger sequencing, and it was heterozygous in the healthy parents and sister. The mutation was classified as Likely Pathogenic according to the ACMG nomenclature (https://varsome.com/ accessed on November 2021).A multigene panel including 271 genes explored by Next Generation Sequencing and involved in hereditary spastic paraparesis, motor neuronopathies, and peripheral nerve involvement see  revealedAltered TBCD protein levels affect microtubule formation leading to abnormal microtubule trafficking in the human brain, and impairing dendrites’ arborization. Microtubule dynamics are complex tightly regulated processes at the basis of cell viability, architecture, and division. Tubulin alpha and beta assemble in heterodimers following the concerted action of chaperone molecules as TBCA to TBCE; in particular, TBCD-β tubulin and TBCE-α tubulin interact with each other, and together with TBCC they form a supercomplex that releases native tubulin heterodimers upon E-site GTP hydrolysis . MoreoveGiven their fundamental role in regulating cell growth and dendrites formation, tubulinopathies are a heterogenous group of conditions characteristically presenting with cortical malformations and dysmorphic basal ganglia ,16. FromHomozygous mutations were also described in several Israeli, Japanese, Faroese, Indian-Jewish, and Egyptian-Jewish children ,9, with TBCD gene (Arg942Gln) was considered to be the causative variant [Interestingly, two siblings were diagnosed with atypical SMA at a very early age during childhood, with hypotonia and muscle weakness indicating lower motor unit dysfunction, and progressive complicated central nervous system dysfunctions, axonal-dominant degeneration or motor unit reduction, and pes equinus of both feet was noted. In this case, two different mutations in two different genes were reported, but the homozygous mutation in  variant .http://genetics.bwh.harvard.edu/pph2/ accessed on November 2021). Upon 3D modeling, we observed that the amino acid change from arginine to glutamine at residue 294 caused a destabilization in the binding site to the complex Arl2/beta tubulin with other residuals in the evolutionary conserved alpha helices structure, altering the physiologic protein function (The mutation found in our case is likely to be harmful, upon in silico prediction using PolyPhen-2 (Polymorphism Phenotyping at function .Our patient presented a milder phenotype compared to most cases reported in the literature, with clinical findings limited to slowly progressive muscle weakness and slight intellectual retardation along with a neurophysiological pattern of axonal-dominant motor neuron degeneration and motor unit reduction . InteresTBCD gene has been only associated with continuous epileptic spasms as a severe form of status epilepticus [In our case, the phenotype was, however, dominated by motor symptoms with neurophysiological evidence of a selective motor neuronopathy at four limbs like SMA. Only recently has the pathogenic link between SMN deficiency and altered microtubule stability been elucidated, pointing to a mitochondrial mislocalization in motor neurons driven by SMN loss . Miyake lepticus , withoutTBCD missense mutations, such as the one here described, c.881G>A, may only partially disrupt the protein capabilities to bind β tubulin-Arl2 and consequently affect tubulin dynamics more selectively in motor neurons compared to cortical neurons during the developmental stages.Therefore, it could be hypothesized that some TBCD gene alterations is complex and growing with the literature data involving mainly neurodevelopment regression and epilepsy. In this context, the molecular basis of these disorders involving the complex tubulin machinery could be better understood through the detection of differential gene variants and their effective clinical impacts.Our case well illustrates how atypical SMA presentations with only mild intellectual impairment should be further inquired for developmental disorders of infancy with central nervous system neuroimaging and extensive genetic panels taking into consideration TBCD and other proteins involved in microtubule dynamics and mitochondrial trafficking. For these selected cases, antisense therapy currently available for SMN1-defective SMA would not be feasible, and other molecules with microtubule-stabilizing functions could be considered. The wide range of neurological effects of TBCD mutations, by the identification of a rare homozygous TBCD variant associated with a predominant distal motoneuronopathy, slight mental retardation, and corpus callosum atrophy, a clinical picture distinct from the literature reports. In this setting, computational modeling methods and accurate genotype–phenotype correlations may better clarify the impact of the secondary and tertiary structure alteration following amino acid substitution, to better understand the mechanism of neurodegeneration related to tubulinopathies, leading to an early diagnosis and appropriate genetic counseling.Our findings extend the phenotypic traits of tubulinopathy generated from