Ancient polyploidization events have had a lasting impact on vertebrate genome structure, organization and function. Some key questions regarding the number of ancient polyploidization events and their timing in relation to the cyclostome-gnathostome divergence have remained contentious. Here we generate de novo long-read-based chromosome-scale genome assemblies for the Japanese lamprey and elephant shark. Using these and other representative genomes and developing algorithms for the probabilistic macrosynteny model, we reconstruct high-resolution proto-vertebrate, proto-cyclostome and proto-gnathostome genomes. Our reconstructions resolve key questions regarding the early evolutionary history of vertebrates. First, cyclostomes diverged from the lineage leading to gnathostomes after a shared tetraploidization (1R) but before a gnathostome-specific tetraploidization (2R). Second, the cyclostome lineage experienced an additional hexaploidization. Third, 2R in the gnathostome lineage was an allotetraploidization event, and biased gene loss from one of the subgenomes shaped the gnathostome genome by giving rise to remarkably conserved microchromosomes. Thus, our reconstructions reveal the major evolutionary events and offer new insights into the origin and evolution of vertebrate genomes. Early vertebrate genomes were shaped by multiple whole-genome duplication (WGD) events of debated timings. Here the authors’ reconstruction of ancestral genomes using the probabilistic macrosynteny model supports a WGD shared by all vertebrates and a gnathostome-specific WGD, and reveals evidence of a cyclostome-specific genome triplication. The common ancestor of vertebrates that originated during the Lower Cambrian1 diverged to give rise to the two extant lineages of vertebrates, the cyclostomes (jawless vertebrates) and gnathostomes (jawed vertebrates). Cyclostomes are a monophyletic group2 comprising lampreys and hagfishes, while gnathostomes include cartilaginous fishes  and bony vertebrates . Cyclostomes are sometimes thought to be morphologically primitive as compared to gnathostomes as they lack hinged jaws, paired appendages and nostrils, mineralized tissue, and a discrete pancreas5. However, recent studies suggest that the lamprey and hagfish lineages independently acquired their seemingly simplified as well as their specialized morphology, and that the ancestral cyclostome already had a complex morphology and physiology distinct from the gnathostome lineage7. For example, although cyclostomes lack the major histocompatibility complex and immunoglobulin-based adaptive immune system (AIS) of gnathostomes, they have independently evolved somatically diversifying variable lymphocyte receptors for antigen recognition8.The emergence of morphologically complex vertebrates from invertebrate chordates is considered a major evolutionary transition that led to the emergence of more than 70,000 vertebrate species  during early vertebrate evolution . However, the timing of 1R and 2R relative to the cyclostome–gnathostome divergence has remained contentious—a significant gap in our knowledge considering the important implications for the genetic basis of the shared and derived features of these two lineages. Previous studies have produced conflicting results supporting each of the three possibilities26, i.e. divergence occurring prior to 1R, between 1R and 2R, and after 2R  created by successive rounds of tetraploidization; lineage-specific secondary losses of some ohnologues15. In particular, this strategy has the potential to reveal chromosome fusion/fission events that occurred in the interval between 1R and 2R and/or after 2R15. Whether such genome rearrangements are shared by cyclostomes and gnathostomes would be potentially informative for determining the timing of the cyclostome–gnathostome divergence in relation to 1R and 2R. A prerequisite for such comparisons is the high-resolution reconstruction of the proto-cyclostome and the proto-gnathostome genomes which require high-quality, chromosome-scale genome assemblies from the most basal vertebrate lineages such as cyclostomes and cartilaginous fishes.An alternative and more effective strategy for the identification of ancient polyploidy is the macrosynteny-based reconstruction of ancestral genomesLethenteron japonicum; also known as the Arctic lamprey Lethenteron camtschaticum) and a cartilaginous fish, the elephant shark , based on long single-molecule reads and chromatin conformation capture (Hi-C) data. These two species represent two crucial divergence points in the evolution of vertebrates 22, presumably due to the higher contiguity of the long-read assemblies. Using the MAKER pipeline (v2.31.8)37 and evidence-based and ab initio gene predictions, we predicted 18,747 protein-coding genes in the elephant shark genome assembly and 19,455 protein-coding genes in the Japanese lamprey genome assembly, respectively.We generated de novo chromosome-scale genome assemblies for elephant shark and Japanese lamprey using a combination of PacBio single-molecule real-time (SMRT) sequencing , and ‘Chicago’34 and comparing the Japanese lamprey, sea lamprey (Petromyzon marinus)19, amphioxus (Branchiostoma floridae)14, and four gnathostome genomes including human, chicken38, spotted gar39 and elephant shark (see ‘Methods'). In our reconstruction procedure, the lamprey genomes were partitioned into segments of conserved macrosynteny (191 Japanese lamprey segments and 198 sea lamprey segments), where synteny breakpoints were detected using the Japanese lamprey, sea lamprey, elephant shark, spotted gar, chicken and human genomes. Then, our Bayesian inference algorithm reconstructed the proto-vertebrate genome, assuming that individual proto-vertebrate chromosomes (Pvcs) have distinct orthologue distributions over the lamprey segments34. The reconstructed proto-vertebrate genome comprises 18 putative chromosomes  and is largely consistent with previous reconstructions with 17 putative chromosomes15  into 104 proto-cyclostome chromosomes (see ‘Methods' for details). Our algorithm enumerates possible combinations of lamprey segments  are unlikely to be missing entirely; therefore, our reconstruction is particularly reliable for the largest five proto-vertebrate chromosomes , which consistently exhibited a multiplicity of six. Thus, the high coverage (60.3%) of the Japanese lamprey genome by sixfold duplicated proto-cyclostome chromosomes suggests that extant cyclostome genomes are paleo-dodecaploids (i.e. the chromosome number increased as 18 × 6 due to tetraplodization and hexaploidization), which might be similar to the situation in sturgeon where a species (Acipenser brevirostrum) with ∼180 chromosomes is considered to be a hexaploid of a tetraploid ancestor with ∼60 chromosomes48.Although the current lamprey genomes might still be incomplete and some chromosomes might be fragmented, such limitations are unlikely to have substantially biased our analysis. First, if the proto-cyclostome genome was shaped by three rounds of tetraploidization, that would additionally require a large number of subsequent chromosome fusions to explain the current genome arrangement . However, we found that the lamprey lineage had remarkably low rates of inter-chromosomal rearrangement  and lacked representatives of its sister group, the cartilaginous fishes (Chondrichthyes). Here, we produced a substantially improved reconstruction of the proto-gnathostome genome structure with a higher coverage of modern genomes by taking advantage of our newly sequenced, chromosome-scale genome assembly of the elephant shark, in addition to the spotted gar, zebra finch, turkey, chicken, opossum, dog, mouse and human genomes  if chromosomal features characteristic to modern avian microchromosomes  were already present in the ancestral gnathostome genome , and (2) why microchromosomes have been conserved in distantly related gnathostome species such as the chicken, spotted gar and elephant shark.Analysis of this proto-gnathostome genome also revealed the origin of microchromosomes found in some modern gnathostomes. Microchromosomes are tiny chromosomes , characterized by high GC-content, high gene density and high recombination rate53. Furthermore, we observed that specific sequence features  are shared by modern gnathostome chromosomal regions that were derived from such proto-gnathostome chromosomes  comprising long-range interacting regulatory elements and/or topologically associating domains (TADs) that require long-range linkage to be maintained intact. To test this possibility we analysed the density of GRBs and TADs54 and observed no obvious difference between macrochromosomes and microchromosomes  remains an unresolved issue in the field of vertebrate genome evolution. In order to resolve the divergence timing conclusively, we searched our reconstructions of the proto-vertebrate, proto-cyclostome and proto-gnathostome genomes for evidence of large-scale genomic changes that help distinguish between three alternative divergence models, i.e., divergence before 1R, between 1R and 2R, or after 2R. Our reconstructions revealed nine major fusion events that occurred during the interval between 1R and 2R  differential gene loss between cyclostome and gnathostome lineages29, (3) delayed rediploidization32 creating cyclostome-specific paralogues between proto-cyclostome chromosomes duplicated by 1R, and (4) tetraploidization through hybridization and doubling59, which may have created both vertebrate-specific and cyclostome-specific paralogues due to recurrent hybridization among genetically diverse subpopulations58 and subsequent genetic drift60. Although these factors may have obscured the duplication timing, the presence of chromosome pairs enriched either with vertebrate-specific paralogues or cyclostome-specific paralogues are consistent with the model that the proto-cyclostome lineage diverged from the proto-gnathostome lineage shortly after 1R.In order to verify the timing of duplications and the gnathostome–cyclostome divergence, we performed a gene tree analysis by inserting lamprey genes into Ensembl gene trees or re-computing the gene trees  Fourth, we present evidence that there was a cyclostome-specific hexaploidization 46, and in the Prussian carp (Carassius gibelio)69. In most instances the mechanism of hexaploidy origin has been inferred to have been by serial hybridizations66.To the best of our knowledge our reconstruction is the first reported genome-scale evidence for hexaploidy in the cyclostome lineage. There are several documented examples of hexaploidy giving rise to new evolutionary lineages. Perhaps the most well-known example is wheat, a domesticated crop with three subgenomes . The formation of hexaploid wheat is believed to be a multi-step process where there was an initial tetraploid genome formed by hybridization, and a subsequent hybridization of the tetraploid with a diploid, generating a hexaploid70. A few possible mechanisms have been suggested for explaining the establishment of allopolyploid species. First, heterosis, or hybrid vigour, confers selective advantages to the newly formed allopolyploids72. Second, asymmetric and unequal contribution from the subgenomes has been reported to have facilitated the evolution of complex phenotypes in some allopolyploids: for example, it was suggested that the allotetraploid cotton produces high-quality fibres by combination of long fibres from the A-genome and short fibres from the D-genome73; in the allohexaploid wheat, the A-genome is responsible for the morphological traits, while the B- and D-genomes contain most genes for response to biotic and abiotic factors74. In line with this argument, previous studies have shown an example of asymmetric contribution from quadruple paralogous regions in the human genome75. Our reconstruction suggests that such asymmetric evolution may not be limited to specific gene clusters but is a genome-scale phenomenon due to the hybrid origin of the allopolyploid proto-gnathostome genome.These instances suggest that genome hybridization may have played a significant role in the origin and evolution of early vertebrates, as already discussed in previous studies77. The seemingly abrupt emergence of such a complex molecular machinery of AIS has been described as an evolutionary ‘Big Bang’ triggered by macroevolutionary events, including the two rounds of tetraploidization80.In particular, our reconstruction suggests that genome hybridization might have contributed to the origin of the adaptive immune system (AIS), which is a prime example of a major evolutionary innovation in early vertebrates. The human AIS is an intricate defence system characterized by the B cell and T cell receptors and the major histocompatibility complex (MHC), which are highly conserved throughout most gnathostomes, including cartilaginous fishes, but are missing in invertebrates, including the closest relatives of vertebrates, such as sea squirts and amphioxus81. However, our reconstruction shows that the hypothesized proto-MHC chromosome was an artefact due to inter 1R–2R chromosome fusions and additional rearrangements in the mammalian lineages. In our reconstruction, the putative proto-MHC chromosome is divided into several proto-vertebrate chromosomes  that are clearly separated in the lamprey, amphioxus and scallop genomes  and leucocyte receptor complex (LRC), which are essential for the mammalian AIS. It has been proposed that the precursors of MHC, NKC and LRC were physically linked on the proto-MHC chromosome, and the tight linkage facilitated co-evolution of highly polymorphic receptors and ligands, giving rise to a putative ‘immune supercomplex’ that was subsequently fragmented as MHC, NKC and LRC in the human genomecis after 2R, suggesting that interacting gene clusters were preserved despite extensive gene loss83. In addition, we observed functional biases between the two subgenomes: the human genes in the segment derived from the shorter subgenome were enriched with ‘defense/immunity protein’ in PANTHER Protein Class  prioritize copy-number variations in personal genomes for potential pathogenicity.Finally, our reconstruction of the proto-gnathostome genome has implications for understanding the intrinsic evolutionary constraints on gnathostome genomes. It has previously been shown ohnologues are frequently dosage-sensitive and resistant to evolutionary duplication and lossres Fig.  associatIn conclusion, we have generated high-quality, chromosome-scale genome assemblies for two phylogenetically opportune organisms, and inferred the genome structures of early vertebrate lineages. This is the first effort to reconstruct the proto-cyclostome genome, which was critical for determining the cryptic origins of the proto-cyclostome and proto-gnathostome genomes. Consequently, our reconstruction resolved several important issues including the number and relative timings of polyploidization events that occurred during the early origin of vertebrates. The resulting model offers unique perspectives on the origin and evolution of vertebrate genomes.34, which was previously used for inferring the structure of the pre-TGD genome (TGD stands for teleost-specific genome duplication). The details including the probability model, definitions of parameters/variables, algorithm and estimation accuracy can be found in ref. 34 (open access). In short, the macrosynteny model assumes that the individual pre-WGD chromosomes have distinct orthologue distributions over the present-day post-WGD genomes; then, the pre-WGD genome structure can be reconstructed by employing the variational Bayesian inference algorithm. In the present study, we used an algorithm called collapsed variational Bayes (CVB)89, which is more efficient than the variational Bayesian expectation-maximization algorithm described in ref. 34.We reconstructed the proto-vertebrate genome by employing the probabilistic macrosynteny model34. In the framework of the probabilistic macrosynteny model, we infer the pre-WGD genome structure as the posterior  algorithm89 for the probabilistic topic model91, we obtain Algorithm 1 with the following update formula:34 and C is a constant that cancels by normalizing q′ denotes the estimate in the previous iteration.The CVB algorithm is derived as follows using the same model and variables as defined in ref. Below is a pseudocode for the CVB0 algorithm.We reconstructed the structure of the proto-vertebrate genome in two steps: first, we partitioned the lamprey genomes into blocks of conserved synteny by comparing the lamprey genomes with each other and also with four gnathostome genomes ; second, we inferred the structure of the pre-WGD genome by applying the macrosynteny model to the amphioxus and lamprey genomes. These steps are described below.34. Specifically, we employed the Bayesian segmentation model92 and computed the optimal segmentation using a dynamic programming algorithm93. Segmentation was performed in two steps: first, we compared the Japanese lamprey and sea lamprey scaffolds, and identified lineage-specific synteny breakpoints; second, we compared the lamprey genomes with human, chicken, spotted gar and elephant shark genomes, and identified breakpoints occurring between the gnathostomes and cyclostomes. Then we merged the two sets of breakpoints and obtained 191 Japanese lamprey segments and 198 sea lamprey segments. These segments have homogeneous distributions of orthologues in the other genomes under comparison, and thus they are likely to have been unaffected by large-scale inter-chromosomal rearrangements in the cyclostome lineages.We partitioned the lamprey scaffolds (with at least ten genes) into blocks of conserved synteny as described in ref. 14 and the Japanese lamprey and sea lamprey segments, by applying the macrosynteny model and CVB0 algorithm with the following parameter values: post-WGD species 34 for details of these parameters). As described in ref. 34, individual amphioxus scaffolds were associated with mixture distributions over the proto-vertebrate chromosomes, which represent reconstruction confidence scores. For the sake of simplicity in visualization, we assigned each amphioxus scaffold to the proto-vertebrate chromosome with the largest reconstruction confidence score ; then, the paralogue distribution should be highly non-random, with most paralogues found between lamprey segment pairs both deriving from the same proto-vertebrate chromosome. We quantified such non-randomness by using the hypergeometric distribution under the null hypothesis in which paralogues are randomly distributed over the entire genome as described below.The underlying assumption is that most lamprey paralogues were created by WGDs , which is computationally infeasible because the number of all set partitions, known as the Bell number, can be extremely large: for example, the Bell number for the 21 lamprey segments is Step 1: Paralogous lamprey segments do not originate from the same proto-cyclostome chromosome. Therefore, we calculated the paralogue significance for each segment pair, and significant pairs were not allowed to be assigned to the same cluster in the subsequent steps.Step 2: Orthologous segments between Japanese lamprey and sea lamprey originate from the same proto-cyclostome chromosome. Therefore, we performed a single linkage clustering of lamprey segments to make clusters of orthologous segments. First, we defined individual segments as initial clusters. Second, we sorted segment pairs by the significance of the number of orthologues between them. Third, we repeated choosing the most significant segment pair and merged the two clusters if they did not have paralogous segments.Step 3: Some lamprey scaffolds are expected to be over-fragmented by the synteny segmentation algorithm or by lineage-specific rearrangements. In order to address such over-fragmentation of lamprey scaffolds, we merged two clusters if (i) they had segments on the same scaffold and (ii) they did not have paralogous segments.Step 4: In addition to Step 3, we utilized the Pacific lamprey linkage markers19 and merged two clusters if (i) the clusters shared a pair of sea lamprey segments having linkage markers on the same Pacific lamprey linkage group and (ii) the clusters did not have paralogous segments.Step 5: Reliable reconstruction is difficult for short segments having few orthologues and paralogues. Therefore, clusters of lamprey segments were excluded from the proto-cyclostome reconstruction if the clusters had fewer than five genes.For each of Pvc1–Pvc17, we enumerated all set partitions of the clusters, and chose the optimal set partition with the most significant distribution of orthologues and paralogues as the proto-cyclostome chromosomes. During this analysis we found that some Japanese lamprey scaffolds are likely to be haplotype sequences that were not removed from the primary assembly by FALCON during the final stage of the assembly; we therefore excluded the following Japanese lamprey scaffolds from the proto-cyclostome reconstruction: Scaffolds 110, 190, 198, 105, 104, 82, 163, 69, 133, 74, 115, 139, 86, 70, 72, 171 and 192. We left Pvc18 as a single proto-cyclostome chromosome because computation of the optimal set partitioning was infeasible for Pvc18 consisting of 34 segments.Significance of paralogues, orthologues and set partitions were calculated as follows.The significance of the number of paralogues between two lamprey segments is calculated as follows. Let The significance of the number of orthologues between two lamprey segments is calculated as follows. Let For each proto-vertebrate chromosome In addition, we calculated the significance of the number of orthologues between species c asFinally, we defined the significance of the set partition for proto-vertebrate chromosome 13, which was developed for verifying if genome quadruplication occurred in the proto-vertebrate lineage: in the previous study, set partitioning into 2, 3, 4 and 5 post-2R chromosomes were enumerated for showing that quadruplication was the most significant; we extended it to also enumerating set partitions into more than five proto-cyclostome chromosomes.This method is an extension of the reconstruction of post-2R chromosomes in ref. 14 and several gnathostome genomes including elephant shark. As illustrated in Fig. We reconstructed the proto-gnathostome chromosomes by comparing the amphioxus34. Specifically, we performed genome segmentation twice for each gnathostome genome: one with four teleost genomes  to identify blocks of doubly conserved synteny, and the other with chicken, turkey, zebra finch, anole lizard and spotted gar to identify additional synteny breakpoints in individual lineages. Then we merged the two sets of synteny breakpoints to define 151 human segments. Similarly, we partitioned the mouse, dog, opossum, chicken, turkey, zebra finch, spotted gar and elephant shark genomes into 258, 212, 163, 70, 69, 60, 78 and 132 segments, respectively. These numbers of segments are slightly different from the previous study34, because we used the spotted gar genome in addition to the non-mammalian amniotes in the synteny segmentation step.We partitioned the gnathostome genomes as described in ref. 13. However, as we argue in Fig. We used the macrosynteny model and applied the CVB0 algorithm with the following parameter values: number of post-WGD species In this step, we used segments from the human, mouse, dog, opossum, chicken, turkey, zebra finch, spotted gar and elephant shark genomes, and enumerated set partitions for each of the 10 groups after reducing the number of set partitions by clustering of gnathostome segments as in the reconstruction of proto-cyclostome chromosomes:Step 1: Paralogous gnathostome segments do not originate from the same proto-gnathostome chromosome. Therefore, we calculated the paralogue significance for each segment pair, and significant pairs were not allowed to be assigned to the same cluster in the subsequent steps.Step 2: Orthologous gnathostome segments originate from the same proto-gnathostome chromosome. Therefore, we performed a single linkage clustering of gnathostome segments to make clusters of orthologous segments. First, we defined individual segments as initial clusters. Second, we sorted segment pairs by the significance of the number of orthologues between them. Third, we repeated choosing the most significant segment pair and merged the two clusters if they did not have paralogous segments.Step 3: Reliable reconstruction is difficult for short segments having few orthologues and paralogues. Therefore, clusters of gnathostome segments were excluded from the proto-gnathostome reconstruction if the clusters had fewer than five genes.13. Figure For the reconstruction of proto-gnathostome chromosomes, we assumed that individual gnathostome segments might have derived from multiple proto-vertebrate chromosomes, since it was reported that several rearrangements occurred between the two WGD eventsThen, for each cluster c by multiplying the significance values for all species and all species pairs:Significance of orthologues between two gnathostome species was given in the same way as in the proto-cyclostome reconstruction. Then, we calculated the significance of a set partition for cluster In the last step of the proto-gnathostome reconstruction, we chose the most significant set partition and filtered out small unreliable subgroups. This filtering step was necessary because reliable subgroups are expected to have segments from all (or most) of the gnathostome species, whereas reconstruction errors result in spurious small subgroups having short segments from few species. For this reason, we filtered out small subgroups with segments from only few (<3) species, which filtered out nine small subgroups consisting of four mouse segments, one chicken segment and nine elephant shark segments. This filtering step should have little influence on the analysis results because the number of genes on the filtered segments is only 288 in total. Finally, the remaining subgroups were defined as the proto-gnathostome chromosomes.Here, we introduce a framework for calculating the probability that multiplicities of independently duplicating chromosomes converge toward a given ploidy level, where the convergence is measured in terms of the deviation (Let us consider the following situation. The proto-vertebrate genome with The desired probability is calculated as follows. First, the total number of duplication scenarios is given by In our reconstruction, we have Supplementary Table Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Reporting Summary


Accumulated evidence suggest that the adverse outcome of severe coronavirus disease 2019 (COVID-19) is closely related to prothrombotic microvascular pathology with a high risk of venous thromboembolism. Furthermore, the first observational studies indicated that adjunct therapy with low-molecular weight heparin (LMWH) was associated with lower mortality in this cohort of patients. However, the timing of starting LMWH and the dose remain controversial in COVID-19 patients. Considering the above, the aim of this study was to reveal the rationale for using LMWH in the therapy of symptomatic COVID-19 patients based on experimental and clinical studies on LMWH in inflammatory settings with special consideration given to randomized trials. In Several mechanisms have been proposed that may lead to cardiac microcirculatory dysfunction in the course of viral infections: (1) a persistent inflammatory response, which can beget local thrombosis, (2) neurohormonal activation including elevated levels of catecholamines, and (3) tissue hypoperfusion with subsequent development of oxidative stress ,17,18. CAnticoagulant therapy with low-molecular weight heparin (LMWH) reduced the incidence of thrombotic complications without an increase in bleeding events and was associated with lower mortality in seriously ill COVID-19 patients . However1.1in vitro inhibition of viruses using heparin [in vitro experiments showed that heparin could inhibit proinflammatory cytokine gene expression by lipopolysaccharide-stimulated human mononuclear cells [Since 1964, a number of studies have reported the  heparin ,24. Furt heparin ,26. Thes heparin ,28. Morear cells ,30. Furtar cells . In addiar cells . Heparinar cells . Apart far cells .1.2The first observational studies indicated that COVID-19 patients treated with LMWH had a better survival rate . TherefoSearching for answers to these questions, it is worth analyzing two randomized trials on LMWH including HF patients in whom inflammatory settings with procoagulant conditions were evident, and the viruses were involved in its development ,37,38,39The first randomized trial by De Lorenzo et al. aimed to determine whether a short-term therapy with LMWH was safe in patients with HF . FurtherAs opposed to the trial by De Lorenzo et al., our study was mainly related to the assessment of the efficacy of therapeutic doses of LMWH (enoxaparin) in a long-term therapy (3 months) and long-term (12 months) maintenance of potential improvement in a similar cohort of HF patients. The first randomized trial with therapeutic doses of LMWH in HF patients was published in 2006 . One-hunApart from that, the long-term LMWH therapy resulted in a sustained beneficial effect on cardiac function without deleterious effects on major cardiac events in such a cohort of patients. In several patients, right ventricular endomyocardial biopsy was performed, which suggested the effectiveness of LMWH in the therapy of inflammatory conditions . Our finBased on these observations and their extrapolation to COVID-19 patients, it seems to be rational to introduce therapeutic doses of LMWH immediately after the first occurrence of symptoms and follow such an approach for at least 10 weeks in outpatient settings.2In most cases, the SARS-CoV-2 infection affects highly vascularized organs, including the lungs, heart, and kidney. In addition, a significantly higher mortality is reported in patients infected with SARS-CoV-2 who are elderly, diabetic, hypertensive, or have any other cardiovascular disease. Importantly, all these pathologies are characterized by their dysfunctional microcirculation with hypercoagulability. As a result, the SARS-CoV-2 infection facilitates inflammatory microvascular dysfunction and diffuse intravascular coagulation in the already existing pathology. Anticoagulant therapy with LMWH was associated with lower mortality and reduced the incidence of thrombotic complications without an increase in bleeding events in seriously ill COVID-19 patients. These findings seem to indicate that the use of LMWH can improve the microcirculatory function in patients with pre-existing endothelial dysfunction. It may be suggested that adjunct long-term therapeutic doses of LMWH may be beneficial for COVID-19 patients without deleterious effects in a long-term therapy. We believe that the extrapolation of the results of these two trials to severely ill COVID-19 patients might be useful in the decision-making process. In our opinion, patients hospitalized due to COVID-19 should receive therapeutic doses of LMWH immediately after hospitalization, which should be continued for a long time in the outpatient setting (based on our experience of at least 10 weeks).


Four RCTs were retained for analysis out of 285 eligible articles. MD in haemoglobin concentration between patients with pre-operative iron and patients without pre-operative iron was of 0.81 g/dl . Pre-operative iron did not lead to reduction in the incidence of peri-operative blood transfusion in terms of RD  or RR . To conclude, pre-operative iron significantly increases haemoglobin concentration by 0.81 g/dl before abdominal surgery but does not reduce the need for peri-operative blood transfusion. Important heterogeneity exists between existing RCTs in terms of populations and interventions. Future trials should target patients suffering from iron-deficiency anaemia and assess the effect of intervention on anaemia-related complications.Professional surgical societies recommend the identification and treatment of pre-operative anaemia in patients scheduled for abdominal surgery. Our aim was to determine if pre-operative iron allows correction of haemoglobin concentration and decreased incidence of peri-operative blood transfusion in patients undergoing major abdominal surgery. MEDLINE, Embase and CENTRAL were searched for RCTs written in English and assessing the effect of pre-operative iron on the incidence of peri-operative allogeneic blood transfusion in patients undergoing major abdominal surgery. Pooled relative risk (RR), risk difference (RD) and mean difference (MD) were obtained using models with random effects. Heterogeneity was assessed using the Q-test and quantified using the I In patients undergoing colorectal surgery, study of the NSQIP database revealed that 47.4% of patients were suffering from anaemia at the time of surgical admission2. The aetiology of anaemia is diverse, and the NHANES III study including 2,814,000 participants showed that about one third of anaemia cases were caused by nutritional deficiencies3. In the context of gastrointestinal surgery, this includes anaemia caused by occult bleeding in patients suffering from cancer of the gastrointestinal tract.The global prevalence of anaemia was estimated to be 32.9% according to data from the Global Burden of Diseases, Injuries and Risk Factors 2010 Study4, increased morbidity5, increased mortality6 and prolonged length of hospital stay5.Pre-operative anaemia might reflect advanced cancer or poor health status, but can also lead to impaired oxygen delivery to tissues and increased morbidity in the perioperative period. For instance, pre-operative anaemia was reported to be associated with increased post-operative incidence of surgical site infection7. Therapeutic interventions include pre- or peri-operative blood transfusion, pre-operative erythropoietin administration11 and pre-operative iron administration. The National Institute for Health and Care Excellence (NICE) NG24 recommendation advises to restrict erythropoietin administration in the surgical setting for anaemic patients refusing blood transfusion or in case of non-compatibility with available transfusion, and to offer oral iron in patients with iron-deficiency anaemia before surgery12.Having considered the potential complications caused by pre-operative anaemia, the Enhanced Recovery After Surgery (ERAS) Society recommends to screen for pre-operative anaemia and to correct it when present16. Therefore, we aimed to determine if pre-operative iron allows a reduction in the incidence of peri-operative blood transfusion in patients undergoing major abdominal surgery and correcting haemoglobin concentration  in the fieldon Table .versus no iron or placebo in patients undergoing abdominal surgery were retained. Non-randomized studies, letters, secondary analyses of original studies, RCT protocols and trials not reporting the incidence of peri- or post-operative allogeneic blood transfusion in the interventional and control groups were excluded. Two independent reviewers  performed the literature screening. In case of disagreement, consensus was reached with a third author (RJD). Pooled relative risk (RR), risk difference (RD) and mean difference (MD) were obtained using models with random effects. Heterogeneity was assessed using the Q-test and quantified using the I2 value. Risk of bias was assessed using the RoB2 Cochrane Collaboration’s tool for assessing risk of bias in RCTs17. Publication bias was investigated using funnel plots18. The software Review Manager  was used for the meta-analysis and the risk of bias assessment. The systematic review and meta-analysis complied with the PRISMA guidelines19 .MEDLINE, Embase and CENTRAL were searched without time limit to 28.12.2020 for RCTs written in English assessing the effect of preoperative iron administration on the incidence of allogeneic blood transfusion (primary outcome) and correction of anaemia (secondary outcome) during and after abdominal surgery Table . Referen19 Table , respect16 were included for analysis  in favour of patients who received pre-operative iron 16 reported the number of patients who received blood transfusion in both the intervention group and the control group. The risk difference in terms of blood transfusion was not significantly reduced by 13 percentage points  in patients who received pre-operative iron when compared to patients who did not receive pre-operative iron   in favour of iron, with a pooled result becoming statistically significant and with reduced heterogeneity. Similarly, the RR was of 0.41 .The four included RCTs (651 patients)21. However, no subgroup analysis was performed for patient who underwent abdominal surgery, and the effect on blood transfusion was not reported by another meta-analysis pooling only RCTs22. The population of patients undergoing abdominal surgery was specifically explored by a systematic review, which concluded that preoperative iron allowed increasing haemoglobin concentration, but did not allow decreasing the incidence of allogeneic blood transfusion23. The FAIRY trial also showed that haemoglobin concentration could also be increased by post-operative administration of iron in anaemic patients after gastrectomy24.The beneficial effect of preoperative iron on haemoglobin concentration and allogeneic blood transfusion was reported by a recent systematic review and meta-analysis pooling data from different surgical specialtiesIn the present systematic review and meta-analysis pooling only RCTs and specifically including patients undergoing abdominal surgery, we showed that administration of pre-operative iron allowed increasing haemoglobin concentration by 0.81 g/dl at time of admission.16 was excluded from the pooled analysis. In this case, heterogeneity of the results decreased from 64 to 0%. However, the trial by Richards et al. is also the one with the biggest sample size and no reason exists for excluding it from the pooled analysis.However, no significant effect of pre-operative iron was found in terms of perioperative allogeneic blood transfusion, unless the latest RCT by Richards et al.16 out of the four included RCT, including the RCT by Richard et al., did not specifically include patients suffering from iron-deficiency anaemia, therefore preventing us from performing the planned sensitivity analysis based on the presence or absence of iron-deficiency anaemia. As we have previously commented in relation to the PREVENTT trial25, this might lead to statistical underpowering of these trials in the evaluation of the effect of pre-operative iron. In addition, Lidder et al. included a total of only six anaemic patients in the intervention group and 14 in the control group. Considering that iron-deficiency anaemia represents about one third of anaemia causes3, the iron intervention might only have an effect on two patients in the intervention group, which is far too small to show any potential effect of pre-operative iron even in case of an extremely efficient treatment14. Moreover, five patients belonging to the control group received IV iron.By looking closely at the PICO questions of existing RCTs in the field, as summarized in Table 15. Froessler et al. reported a sample size calculation including 134 patients per group13. However, in the published article, only 72 patients were included: 40 in the intervention group and 32 in the control group. Nevertheless, it appears that the trial was terminated earlier than expected due to poorer outcome in the control group. Therefore, when targeting only patients with iron-deficiency anaemia, an effect of pre-operative iron on the incidence of peri-operative transfusion can be noticeable even with low numbers of patients.Included trials were also underpowered when comparing the total numbers of patients included with the numbers of patients initially planned. For instance, Edwards et al. included 60 patients in their final analysis and showed no significant effect of pre-operative iron, but their initial study protocol registered into the EU clinical trials register (2005-003608-13) indicated 126 patients12, which recommend administering pre-operative iron specifically in patients suffering from iron-deficiency anaemia, and would be preferable in terms of patient safety (patients without iron-deficiency anaemia should not receive intravenous iron) and health economics.Considering the limitations of existing RCTs, we recommend that future trials only include anaemic patients with iron-deficiency or, in case of a pragmatic approach including all anaemic patients, to at least perform the sample size calculation based on patients with iron-deficiency anaemia. This would be in line with the NICE guideline15, 4–21 days13 or 10–42 days16 before surgery, although haemoglobin concentration was shown to increase over time after administration of iron26. Moreover, we note that a recent prospective observational study including 1′728 surgical patients showed that iron supplementation allowed to decrease the incidence of post-operative blood transfusion in iron-deficient patients only if it was given more than 7 days before the surgery27, which corresponds to the time required for erythropoiesis. In addition, 3 RCTs administered intravenous iron16 and one provided oral iron14. Although intravenous iron was shown to be more effective in correcting iron-deficiency anaemia in abdominal surgery, no difference could be found between intravenous and oral iron in terms of peri-operative blood transfusion, notably by the IVICA trial29.Another potential source of heterogeneity was the timing and mode of administration of pre-operative iron, which raised some concerns regarding efficiency of trials interventions. For instance, in included RCTs, iron was given 2 weeks30 unless explicitly specified in the trial protocol. None of the included trials used strict criteria for blood transfusion, which was most often the result of decision of the anaesthetic team15. Further, peri-operative blood transfusion is the treatment of anaemia and not a consequence of poor tissue perfusion and hypoxia. Peri-operative blood transfusion might therefore constitute a confounding factor of the effects of anaemia, and measuring directly the effects of anaemia to assess the efficiency of the trial intervention (iron) might potentially show an effect of that intervention.We also noted that included RCT reported the incidence of peri-operative blood transfusion as the main or secondary outcome to measure the efficiency of pre-operative iron on the prevention of anaemia-related complications. We believe that the indication for peri-operative blood transfusion based on haemoglobin concentration is subject to heterogeneityFinally, we note that 3 out of the 4 RCTs included in the meta-analysis were considered to be of high risk of bias, as assessed by the RoB2 Cochrane Collaboration tool.31, therefore questioning about the choice of outcomes to assess the potential beneficial effect of administration of iron. An analysis of the findings of our meta-analysis  in the light of other outcomes than peri-operative blood transfusion would be of interest.Therefore, we think that future RCTs in the field should also report the incidence of anaemia-related complications in the intervention and in the control groups. In abdominal surgery, this should include reporting the incidence of surgical site infection and anastomotic leak. In this regards, it is noteworthy to mention that the long-term follow-up of the IVICA trial reported that patients with colorectal cancer who responded to correction of pre-operative anaemia had improved 5-year overall survival compared with patients who did not respond to iron32 should target patients suffering from iron-deficiency anaemia and assess the effect of intervention on anaemia-related complications.In conclusion, pre-operative iron significantly increases haemoglobin concentration by 0.81 g/dl (8.1 g/l) before abdominal surgery but does not reduce the need for peri-operative blood transfusion. Important heterogeneity exists between available RCTs in terms of populations and interventions. Future trials in the fieldSupplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.


In a bivariate analysis that was adjusted for age, DMEK was associated with a smaller decrease in posterior MK at the 1-month , 3-month , 6-month  and 12-month  follow-up visits. There were no significant differences in changes in anterior or combined surface MK throughout the observation period. Accordingly, no significant differences in changes in MA, AA or HOAs compared to the baseline values were identified between the eyes after DMEK and UT-DSAEK at any follow-up time point. Conclusions: UT-DSAEK seemed to be an easier and safer technique than DMEK while maintaining similar outcomes regarding irregular astigmatism and total keratometry values.Background: The need to provide a comparative analysis of corneal parameter changes compared to their preoperative values between Descemet membrane endothelial keratoplasty (DMEK) and ultrathin Descemet stripping automated endothelial keratoplasty (UT-DSAEK) patients. Methods: The study included 24 eyes after UT-DSAEK and 24 eyes after DMEK. Visual acuity, endothelial cell count (ECC), central corneal thickness (CCT), mean keratometry (MK), mean astigmatism (MA), astigmatism asymmetry (AA) and higher-order aberrations (HOAs) were assessed at baseline and 1, 3, 6 and 12 months after the surgery. Results: From the 3rd month post operation, ECC was higher in the DMEK eyes than in the UT-DSAEK eyes ( The introduction of endothelial keratoplasty (EK) has revolutionized corneal transplantation over almost the past two decades. Descemet stripping automated endothelial keratoplasty (DSAEK), which is a procedure that involves the selective removal of the dysfunctional endothelium, followed by the transplantation of donor corneal endothelium, Descemet membrane (DM) and a portion of donor corneal stroma, replaced penetrating keratoplasty (PK). Accordingly, Descemet membrane endothelial keratoplasty (DMEK), comprising the selective transplantation of a donor button that is composed of endothelium and DM without the posterior stroma, replaced DSAEK as the most common type of corneal transplantation ,2.In 2013, Busin et al. pointed out that ultrathin Descemet stripping automated endothelial keratoplasty (UT-DSAEK) might be a procedure that shares the improved visual outcome and lower immunologic rejection rate of DMEK over DSAEK while minimizing all types of postoperative complications. It was also highlighted that, similar to DSAEK and unlike DMEK, UT-DSAEK can be performed for all types of eyes, even those with complicated anatomy or poor anterior chamber visualization [There are only a few studies that compared DMEK and UT-DSAEK, and they mainly focused on visual acuity outcomes, contrast sensitivity, endothelial cell loss or complication rates ,7,8,9,10Only a few studies have compared higher-order aberrations, astigmatism, corneal pachymetry and keratometry using either Schleimpfung cameras or tomographs with corneal topographers between eyes that have undergone either DMEK or UT-DSAEK. The concomitantly growing popularity of anterior segment optical coherent tomography (AS-OCT) has caused the use of this device in clinical practice to become increasingly frequent. To the best of our knowledge, to date, there have been no studies that compared corneal topographic parameters between eyes that underwent DMEK and UT-DSAEK in swept-source AS-OCT. Bearing in mind the above, our aim was to create a study to provide an analysis of corneal parameter changes in patients who underwent DMEK and UT-DSAEK using an AS-OCT device.This study included 24 consecutive eyes that had undergone UT-DSAEK and 24 consecutive eyes that had undergone DMEK due to various causes of endothelial decompensation . The subjects that were enrolled in the study were patients that were operated on at the First Ophthalmology Clinic in Szczecin in 2018-2020 and then followed up at 1, 3, 6 and 12 months after the surgery. All participants underwent a complete ophthalmologic examination, including the following: best-corrected distance visual acuity with Snellen charts , slit lamp biomicroscopy, IOP measurements and a detailed fundus examination after pupil mydriasis. Corneal quality parameters were measured with swept-source AS-OCT at each following visit. 2.Donor corneas were obtained with the multiorgan procurement method and in the dissecting room during autopsies. Corneoscleral buttons were hypothermically stored in Eusol-C medium  at 2–6 °C at the West Pomeranian Eye Tissue Bank in Szczecin. The prestorage evaluation of the endothelium was performed using specular microscopy . All corneas had an endothelial cell count of at least 2800 cells/mmEach surgery was performed by the same surgeon. All subjects underwent prophylactic basal laser iridectomy  before endothelial keratoplasty to minimize the risk of postoperative pupillary block. Subjects with retinal diseases that significantly affected visual acuity were excluded from the study. All procedures were performed with peribulbar block.UT-DSAEK grafts were prepared with the single-pass technique using a MORIA One Use microkeratome. The donor tissue was mounted on an artificial anterior chamber (AAC), which maintained a continuous intracameral pressure of 200 mmHg . After ensuring adequate pressure in the AAC system, central corneal thickness measurements were taken using an ultrasound iPac Pachymeter . An appropriate microkeratome head was chosen, and the cut was performed at a deliberately slow speed. The anterior cap was removed, and the residual stromal bed was again measured. The AAC was carefully disassembled, avoiding any trauma to the endothelium.The donor’s rolled endothelial graft was inserted using a Busin glide through a 3.2 mm clear corneal incision . After centering the graft, the anterior chamber was filled with air to allow for a perfect adherence of the donor flap to the receiving tissue.The preparation of the DMEK graft and surgery was performed following the ‘no-touch’ technique, as described previously .All patients were instructed to keep a supine position after the surgery till the control of the flap position was done on the first postoperative day. In the case of a pupillary block or ocular hypertension, topical mydriatics were administered, and if this was insufficient, a small quantity of air was released from the AC in the operating theatre. The postoperative treatment for both groups was a topical antibiotic given 4 times a day for 1 week and topical preservative-free dexamethasone sodium phosphate 8 times a day for the first month. The topical steroid was tapered down to one drop every other day and then discontinued over 1 year. The graft thickness in all UT-DSAEK eyes was below 100 µm (mean graft thickness at the 12-month follow-up visit was 66.09 ± 18.61 µm). Both the corneal thickness and keratometry values were determined using a swept-source anterior segment OCT CASIA2 . During the entire observation period, the CASIA2 was placed in the same room under the same lighting conditions. All measurements were taken by trained operators, who also held the subjects’ eyelids gently to avoid pressure on the globe. The scan was performed using the autoalignment function. The CASIA2 measurements were obtained with the corneal map mode of the anterior segment module from the OCT images using the built-in software and measurement tools provided by the manufacturers. The CASIA software automatically defined the intraocular structures and generated measurement values. The images were analyzed using built-in 2D analysis software that automatically calculated the measurements, along with the structural outlines and reference lines. The outline tracer was edited where needed.Central corneal thickness (μm), mean keratometry values (D), astigmatism power (D) and axis (°), astigmatism asymmetry (D) and higher-order aberration power (D) were recorded and analyzed at different time points after surgery using the Fourier Analysis 3D/2D function. Measurements were read from both the anterior and posterior surfaces of the cornea, and the total values were taken into account. All parameters were assessed in 3 and 6 mm diameter optical zones (OZs). The image quality was assessed during the acquisition by the operator. Only measurements that were well-centered and with high-quality indexes were included in the study.p < 0.05), non-parametric tests were used. The Wilcoxon signed-rank test was used to compare the preoperative and postoperative values. The Mann–Whitney U test was applied to analyze the differences between the groups. The correlations between the baseline variables and the corneal parameters were analyzed with Spearman’s rank correlation coefficient (Rs). Bivariate analysis, including surgical technique and patient’s age as independent variables, and change of a parameter relative to its baseline value as the dependent variable, was performed using a general linear model (GLM). Standardized beta values (β) were presented to show the strength of association between the surgical technique and the follow-up parameter changes. A p-value of less than 0.05 was considered to be statistically significant. The statistical power of the study at the 0.05 significance level with 24 subjects in each study group was sufficient to detect with 80% probability the real effect size corresponding to the difference between the groups equal to ±0.83 SD and associations between variables within groups with a real effect size corresponding to the correlation coefficient of ±0.54 [Statistical analysis was conducted using Statistica software. Since most of the analyzed variables showed distributions that were significantly different from normal distributions  or UT-DSAEK (n = 24). p = 0.55) and the DMEK group was younger than the UT-DSAEK group (p = 0.01). At the baseline, there were no significant differences between the groups in the values of corneal topographic parameters, i.e., the mean keratometry (MK) values, astigmatism power and asymmetry (AA) and the values of higher-order aberrations (HOAs). Accordingly, the groups did not differ in preoperative central corneal thickness measurements. Forty-eight eyes of 43 patients were assigned to DMEK . No further complications in those eyes were observed.Three patients in the UT-DSAEK group were lost at the 12-month follow-up point. No graft failures or rejections were observed in this study. In four eyes after UT-DSAEK and three eyes after DMEK surgery, a postoperative graft detachment was observed. All of them were recorded in the first 24 h after surgery, with one exception: one DMEK graft detachment took place one week after surgery. In this case, the patient admitted to not following the postoperative recommendations. All detached grafts were successfully attached due to intracameral SFp = 0.01). Up to 6 months post operation, the BCVA improved in both treatment groups to a similar extent from baseline. After adjusting for age, the change in BCVA did not differ significantly between DMEK and UT-DSAEK patients at the 1-month follow-up visit , 3-month follow-up visit  or 6-month follow-up visit . In contrast, at the 12-month follow-up visit, the postoperative increase in the BCVA was higher in the DMEK group (median = 1.00) than in the UT-DSAEK group . This difference remained significant in the multivariate analysis that was performed using a GLM after an adjustment for age .The mean baseline BCVA was 0.22 in the DMEK arm and 0.11 in the UT-DSAEK arm . From 3 months post operation, the endothelial cell density was higher in the DMEK eyes than in the UT-DSAEK eyes .Regarding the ECC, we observed no differences in the donor endothelial cell count . For the posterior surface, MK values were lower at all follow-up time points than preoperative values. Interestingly, for the combined corneal surface, there were no differences in MK values between those recorded at 6 and 12 months post operation and the baseline. Contrary to the DMEK group, in the UT-DSEAK group, we observed a significant decrease in MK recordings for both the anterior and posterior corneal surfaces, as well as for total corneas in both the 3.0 and 6.0 mm diameter OZs as early as 1 month post operation (6.0 mm diameter—data not shown). Finally, the combined surface MK values were significantly lower at 12 months post operation than the preoperative values (mean difference = −1.42 ± 1.87 D for the 3 mm OZ).Regarding the astigmatism power, in the DMEK group, we observed a significant decrease for the posterior and total corneas from 3 months post operation and the anterior corneal surface from 6 months post operation. In contrast, in the UT-DSAEK eyes, the astigmatism power remained unchanged until 6 months post operation and decreased for the anterior and total corneas only at 12 months post operation. The magnitude of the posterior astigmatism in the UT-DSAEK eyes remained unchanged throughout the observation period. Interestingly, we found no significant changes in the astigmatism axis at 1, 3, 6 and 12 months after surgery compared to the baseline in either the DMEK or UT-DSAEK group.When analyzing the irregular corneal astigmatism with the asymmetry of astigmatic components and HOAs, we observed similar patterns of decrease in both the UT-DSAEK and DMEK subgroups. At 1, 3, 6 and 12 months after surgery in both investigated groups, the values of the astigmatism asymmetry and HOAs decreased significantly compared to the preoperative values.Throughout the observation period, we found no differences in the values of any parameters for the anterior and total corneas between the DMEK and UT-DSAEK eyes. Importantly, the values of the posterior mean keratometry and the magnitudes of the back astigmatism asymmetry and HOAs were significantly lower in the DMEK group than in the UT-DSAEK group at all follow-up time points. No differences in astigmatism power for either the anterior or posterior region or the total cornea were observed at any time point between the analyzed groups of eyes.p = 0.04) in the multivariate analysis performed using a GLM after an adjustment for age. A multivariate analysis of keratometry changes in the DMEK and UT-DSAEK eyes, after an adjustment for age, revealed that DMEK was an independent variable that was associated with a smaller decrease in posterior mean keratometry at the 1-month , 3-month , 6-month  and 12-month  follow-up visits in both the 3 and 6 mm optical zones. There were no significant differences in changes in anterior surface or combined surface MK between the UT-DSAEK and DMEK groups throughout the observation period. Regarding the astigmatism power, there were no significant differences in the anterior, posterior surface or combined surface astigmatism changes between the UT-DSAEK and DMEK groups, with one exception: posterior astigmatism decreases at the 6- and 12-month follow-up visits were larger in the DMEK group  in the multivariate analysis that was performed using a GLM after an adjustment for age. Accordingly, no significant differences in changes in astigmatism asymmetry and HOAs compared to baseline values were identified between the eyes from the DMEK and UT-DSAEK groups at any follow-up time point.Subsequently, we compared the changes in corneal surface parameters at postoperative follow-up time points relative to the preoperative values between the DMEK and UT-DSAEK groups. Regarding the pachymetry, we found no differences in the CCT changes at the 1-, 3- and 6-month follow-up visits between the study groups. Exclusively, at the 12-month follow-up visit, the decrease in the CCT was larger in the DMEK eyes , 3-month , 6-month  and 12-month  follow-up visits in the DMEK eyes. Analogous correlations were observed in the UT-DSAEK eyes at 1 month , 3 months , 6 months  and 12 months  post operation.Next, we investigated the potential relationships between the corneal parameters and BCVA. We found that the baseline BCVA determined the postoperative corneal thickness at the 12-month follow-up in both the UT-DSAEK and DMEK groups. Accordingly, we observed a positive correlation between the preoperative BCVA and changes in the CCT at the 1-month (Rs = +0.59, To identify the corneal surface aberrations that most affected the BCVA at subsequent postoperative visits, we investigated the associations between the BCVA and topographic characteristics of the cornea in both the DMEK and UT-DSAEK groups . In the We also analyzed the potential associations between the changes in selected corneal parameters at selected time points post operation. We found that decreases in the magnitudes of the astigmatism, HOA and AA values for the total and posterior cornea throughout the observation period, as well as the anterior corneal surface at 6 and 12 months post operation , were poIn recent years, endothelial corneal transplantation has evolved to become the method of choice for the treatment of endothelial damage. Although DMEK seems to offer better and faster visual and refractive results, controversy still exists regarding whether it is a better technique than UT-DSAEK and NT-DSAEK, which are easier techniques and have similar outcomes to DMEK.The results of this study provided a comparison of a wide range of topographic outcomes between DMEK and UT-DSAEK during a 1-year follow-up period. Compared with the UT-DSAEK results, lower mean posterior keratometry, lower posterior astigmatism, lower posterior astigmatism asymmetry and fewer posterior corneal HOAs were found after DMEK. Nevertheless, total corneal parameters were comparable after both procedures.The results of this study indicated significant differences in posterior surface MK values between the keratoplasty techniques, which occurred after surgery. Compared to DMEK, we noted the steeper posterior corneal curvature in the UT-DSAEK group. This finding is consistent with the results of previous studies. Both Torras-Sanvicens et al. and Goldich et al. analyzed the mean anterior and posterior keratometry values and, similarly to us, found significant differences in the posterior keratometry in favor of DMEK ,15. ImpoOn the other hand, hyperopic outcomes tend to occur after posterior lamellar corneal transplantation. This hyperopic shift is very relevant after DSAEK and is described in the literature as ranging from 0.7 to 1.5 D . RegardiIn this study, we demonstrated a significant decrease in astigmatism power in the eyes after DMEK surgery that occurred from 3 months after surgery in the anterior, posterior and total corneas. In contrast, this effect was significantly prolonged in the anterior and total corneas and occurred only after 12 months post operation. We found no changes in posterior astigmatism throughout the observation period in the UT-DSAEK group. This allowed us to assume that the visual rehabilitation of patients after DMEK was shorter than that after UT-DSAEK. Indeed, data provided by Mencucci et al. lead to a similar assumption that postoperative recovery after DMEK might be quicker than that after UT-DSAEK . Our resHOAs may increase after endothelial keratoplasty, which would significantly reduce visual acuity and influence vision quality. Throughout the observation period, we found no differences in the astigmatism asymmetry or HOA values for the anterior and total corneas between the DMEK and UT-DSAEK eyes. Importantly, the posterior absolute values of those parameters were significantly lower in the DMEK group than in the UT-DSAEK group at all follow-up time points. It is noteworthy that previous analyses that compared the changes in HOAs between DMEK and UT-DSAEK showed inconclusive results. In a recent study performed in 2020, Duggan et al. evaluated the outcomes of DMEK vs. UT-DSAEK patients and found that DMEK induced less posterior corneal HOAs than UT-DSAEK. The authors reported that posterior corneal HOA actually increased significantly in the UT-DSAEK group from the baseline to 3 months after surgery and remained significantly higher at 6 and 12 months. The HOAs of the anterior corneal surface in their sample did not differ significantly between DMEK and UT-DSAEK. These results are only partially in line with ours since we documented a similar pattern of decrease in the astigmatism asymmetry and the HOAs of the anterior, posterior and total corneas in both the DMEK and UT-DSAEK groups. Likewise, Mencucci et al. conducted a similar investigation and found that total and posterior corneal HOAs, posterior astigmatism and total coma were significantly lower after DMEK than after UT-DSAEK in both the 4 and 6 mm optical zones . On the Bearing in mind that the presence of HOAs might affect the vision-related quality of life, several research groups have analyzed the objective and subjective visual quality and contrast sensitivity after both procedures. In their cross-sectional, comparative, and observational case series, Torras-Sanvicens et al. conducted an extended and comprehensive analysis that included objective visual quality variables, contrast sensitivity and subjective patient satisfaction, e.g., subjective quality of the surgical technique, level of comfort in the postoperative period, recovery time and preferred eye. The authors found no significant differences in any of the parameters studied between UT-DSAEK and DMEK eyes . AccordiThe results of our study clearly demonstrated that ECC loss after DMEK was less than that after UT-DSAEK. These results are comparable with those published by other research groups . ConversIn conclusion, to the best of our knowledge, this is the first case series that compared changes in corneal topographic parameters with their baseline values between UT-DSAEK and DMEK groups at the 12-month follow-up. Accordingly, this might also be the first study in which the values of corneal topographic parameters in DMEK and UT-DSAEK eyes were studied using CASIA2 swept-source anterior segment OCT. Although DMEK showed a faster recovery during follow-up, along with lower pachymetry and a flatter posterior keratometry than UT DSAEK, UT-DSAEK seemed to be an easier and safer technique with similar outcomes regarding irregular corneal astigmatism with asymmetry of astigmatic components and HOAs.


Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets. At the turn of the 21st century, the field of degradomics emerged as a discipline that employs genomic and proteomic approaches to elucidate protease and protease-substrate repertoires, or “degradomes”, on an organism-wide scale . ConversIn studies investigating the role of proteases in chronic lung disease, significant attention has been given to the serine, cysteine and matrix metallo- (MMPs) protease groups. In particular, extensive findings regarding the role of neutrophil serine proteases (NSPs) in pulmonary disease have been reported . More spThis review provides an overview of serine TLPs before focusing on their role in the activation of ENaC, PAR2 and viruses. Their inhibition and potential as therapeutic targets are also discussed.Proteolytic enzymes are commonly classified according to the reactive amino acid residue which acts as the nucleophile within the catalytic site , or is based on the cofactor necessary for catalytic activity  . Serine A)) are one of the most extensively studied groups of enzymes to date . Pr. Pr52]. via ENaC ,70. Signvia ENaC . Similarvia ENaC .Optimal ASL hydration is also regulated by ENaC-dependent sodium and fluid absorption working in tandem with cystic fibrosis transmembrane conductance regulator (CFTR) chloride secretion across the apical membrane of epithelial cells. Studies carried out to unravel the complexity of CFTR and ENaC interactions have discovered that the absence of CFTR leads to the activation of ENaC by cAMP/PKC (cyclic adenosine monophosphate/protein kinase C), whereas CFTR expression causes ENaC inhibition by cAMP/PKA (protein kinase A) ,74. HencA macromolecular gel-forming mucin known as MUC5AC is a major component of the mucus layer of human airways . MUC5AC Serine proteases have also been implicated in the regulation of various pro-inflammatory signalling pathways through their role in the activation of protease-activated receptors (PARs). As TLP activity has been associated with airway inflammation, PAR2 may not only represent a physiological substrate but also an intriguing pharmacological target in chronic conditions including asthma and COPD.Protease-activated receptor 2 (PAR2) is a member of the G-protein-coupled receptors (GPCRs) super family, along with PAR1, PAR3 and PAR4 . While tq, Gs or G12/13, referred to as “canonical” activation . In contrast, NE displayed no significant associations with lung function or patient survival. This is the first study to highlight a potential role for TLP activity as a novel noninvasive biomarker for long-term risk in CF lung disease.Interestingly, based on the preference of TLPs for an arginine or lysine residue in the P1 position, a number of rationally designed novel compounds have been developed. For example, QUB-TL1 is a synthetic molecule consisting of an arginine-derived diphenyl phosphonate moiety that reacts with the active site serine residues in order to irreversibly bind to TLPs . QUB-TL1To conclude, airway TLPs are an important, yet largely overlooked sub-group of serine proteases. Given the key roles that have already been identified for these enzymes in the regulation of numerous processes involved in airways dehydration, inflammation and viral infection, it seems prudent that further work is conducted to dissect out the contribution made by individual activities. To date, inhibitors, both large and small, have exerted a broad-spectrum activity but have a heightened risk of off-target effects. The development of a more selective approach to inhibition may allow the consideration of the TLPs as potential therapeutic candidates within the context of muco-obstructive lung diseases.


The evolution of the inhomogeneous distribution of the transformation stress ( Superelastic shape-memory alloys (SMAs) are functional materials capable of sustaining a large recoverable deformation strain as a result of a stress-induced martensitic transformation (MT) between austenite and martensite. Among SMAs, Cu-Al-Mn SMAs possess superior features such as low cost, high cold workability, and large transformation strain compared with TiNi-based SMAs . They arRecently, Cu-Al-Mn single crystals with excellent superelasticity have been fabricated using abnormal grain growth (AGG) induced by a cyclic heat treatment ,10. ThesBased on the above-mentioned motivations and the knowledge of large differences in superelasticity properties of the bicrystal Cu-Al-Mn sample , it was 3 and CH3OH (2:8 in volume). The preparation procedures for the bicrystal Cu-Al-Mn sample were detailed in the literature [terature .−3 s−1 such that experiments can be considered as quasi-static. Each compression cycle took about 120 s. A speckle pattern was applied on the observed surface  of the specimen using black and white sprays. The pattern was used for in situ strain tracing and ex post strain field analysis. The deformation strain of the specimen was measured with a virtual strain gauge by optical DIC . Three deformation strains  is higher than the strain rate (2.4 × 10−3 s−1), the deformation behavior of the material can be captured. These snapshots were analyzed ex post in the VIC 3D 8 software to obtain the strain distribution at the surface of the specimen, as shown in the full-field strain measurement in The cyclic compression–unloading test was performed under the strain-controlled mode using a universal tester with a 50 kN load cell . The strain rate used in the compression–unloading test was about 2.4 × 10Based upon the measured results  of the strain fields and the applied loads from the full-field strain measurement, the equilibrated stress distribution at the surface of the specimen can be numerically computed using the DDI method ,15,16 unThe mechanically admissible stress–strain pairs, obtained from the full-field stress and strain measurements shown in d theory ,11,17. Tinset of b. It caninset of . As showorted in . Please Before the beginning of the tenth compression cycle, some regions in the bottom grain had residual strain. At the tenth deformation, the top grain experienced less deformation relative to the first cycle, whereas the bottom grain began to exhibit increased deformation. As shown in At the beginning of the twentieth compression cycle, the band of residual strain at the bottom grain extended, and more residual strain remained. Furthermore, during the loading process, the upper part of the bottom grain showed a level of transformation stress closer to that of the top grain, as shown in By comparing the evolution of the strain distributions during the cyclic deformation, it can be deduced that the region exhibiting a decrease in transformation stress was highly correlated with the region undergoing MT. In the first compression test, the MT band in the bottom grain was clearly identified. During cyclic deformation, the MT in the bottom grain mainly originated from this band, and the residual strain in this region accumulated. This band, which was associated with accumulated permanent deformation, also experienced a more severe decrease in transformation stress than that of the top grain, as shown in To further investigate the relationships between transformation stress  for revealing strain and transformation stress fields, it was found that grain boundary kinking plays an important role in the decrease in transformation stress near the grain boundary in the bicrystal Cu-Al-Mn sample. As can be seen in Furthermore, during the strain-controlled cyclic loading (5% strain for each cycle), the transformation stress of the bottom grain gradually decreased to the values close to those of the top grain b,c. HencThe full-field stress and strain measurements provide a promising technique for measuring stress–strain responses in SMAs. This new method offers not only insights into cyclic superelastic deformation, but also the compressive fatigue behavior of Cu-Al-Mn SMAs. With this method, several future research directions could be considered, including the influence of misorientation between two grains on its mechanical properties, the effect of grain boundary geometry on the grain boundary strength ,40,41, aIn summary, this study investigated the distribution of transformation stress and strain fields in a bicrystal Cu-Al-Mn sample under cyclic compression. The decrease in transformation stress in both grains correlated with the accumulation of residual strain. The accumulation of residual strain depends on factors such as grain orientation along the loading directions, transformation strain, and grain boundaries. These experimental results and analyses demonstrated that dislocation slip and residual martensite were triggered more easily when a higher transformation stress was required to trigger MT, thus resulting in greater residual strain and a larger decrease in transformation stress. The decreasing behavior at the grain boundary was related to strain incompatibility and the angle between loading direction and the normal direction of grain boundary. Consequently, microstructures with low-angle grain boundaries or single crystals, which exhibit more homogenous deformation behaviors and less restrictions from grain boundaries, will demonstrate higher functional stability and thus longer lifetimes during their cyclic service lives.


Laparoscopic total gastrectomy for the treatment of gastric and esophago-gastric junction cancer is on the rise. The procedure’s rate-limiting step is the construction of the esophago-jejunal anastomosis. Most surgeons are performing the anastomosis laparoscopically by utilizing an endoscopic linear or circular stapler; scarce evidence exists regarding laparoscopic hand-sewn esophago-jejunal anastomosis. Herein, we present our technique and results of laparoscopic manual esophago-jejunal anastomosis during totally laparoscopic total gastrectomy for gastric and esophago-gastric junction cancer. Anastomosis was performed in a two-layer fashion. Overall implementation provided excellent surgical outcomes in our cohort of patients; median anastomotic time was 55 min, while median operating time was 240 min. Intra-operative methylene blue leak test was negative in all cases. No anastomotic leak or anastomotic stricture were noted postoperatively. The 30-day and 90-day mortality rates were zero.Laparoscopic total gastrectomy is on the rise. One of the most technically demanding steps of the approach is the construction of esophago-jejunal anastomosis. Several laparoscopic anastomotic techniques have been described, like linear stapler side-to-side or circular stapler end-to-side anastomosis; limited data exist regarding hand-sewn esophago-jejunal anastomosis. The study took place between January 2018 and June 2021. Patients enrolled in this study were adults with proximal gastric or esophago-gastric junction Siewert type III tumors that underwent 3D-assisted laparoscopic total gastrectomy. A hand-sewn esophago-jejunal anastomosis was performed in all cases laparoscopically. Forty consecutive cases were performed during the study period. Median anastomotic suturing time was 55 min, with intra-operative methylene blue leak test being negative in all cases. Median operating time was 240 min, and there were no conversions to open. The anastomotic leak rate and postoperative stricture rate were zero. The 30- and 90-day mortality rates were zero. Laparoscopic manual esophago-jejunal anastomosis utilizing a 3D platform in total gastrectomy for cancer can be performed with excellent outcomes regarding anastomotic leak and stricture rate. This anastomotic approach, although technically challenging, is safe and reproducible, with prominent results that can be disseminated in the surgical community. Gastric cancer (GC) is one of the most common malignancies worldwide and it is the fourth leading cause of cancer-related morbidity . DespiteLaparoscopic total gastrectomy for the treatment of gastric and esophago-gastric junction cancers is on the rise. The first publication in the English literature of laparoscopic surgery for gastric cancer was in 1991 by Ohgami et al., who first reported a laparoscopic wedge resection using a lesion-lifting method . Goh et Technical development of laparoscopic instruments and optics together with the growing surgical experience over time, through benign and malignant upper gastrointestional procedures, have led more surgeons and centers performing complex operations, like laparoscopic gastrectomies for cancer ,7,8. DesWith current evidence, laparoscopic total gastrectomy for proximal gastric and gastroesophageal junction cancer is considered a more advanced procedure compared to open . ClassicThe aim of this study was to describe our technique of laparoscopic hand-sewn esophago-jejunal anastomosis in total gastrectomy for cancer, in a cohort of consecutive cases alongside the evaluation of clinical and histopathological outcomes.Analysis of prospectively collected data regarding 3D-assisted laparoscopic hand-sewn esophago-jejunal anastomosis performed during TLTG for cancer was performed. Operations were performed by a single upper gastrointestinal surgeon (AC) from January 2018 to June 2021. All procedures were identical in terms of surgical team, operating surgeon, patient positioning, surgical approach, extent of lymphadenectomy and EJ anastomosis performed. This anastomotic technique has not been compared to previous ones, such as construction with a linear or circular stapler, due to the low number of the latter methods, which would not provide a sufficient sample for a valid statistical analysis.Study inclusion criteria were adult patients (>18 years old) with no upper age limit and proximal gastric or gastroesophageal junction Siewert type III cancers that were treated with TLTG. Patients under 18 years old or gastroesophageal junction Siewert type I–II cancers were excluded. All operations for benign disease  and all emergency operations were also excluded from the study.Staging was based on clinical assessment, upper gastrointestinal endoscopy with biopsies, computed tomography of the chest and abdomen as well as staging laparoscopy. Computed tomography positron-emission tomography (PET-CT) was selectively performed in esophago-gastric junction cancers. Treatment strategies for all patients were discussed and agreed to in a dedicated multidisciplinary team setting involving surgeons, radiologists, pathologists and medical oncologists.The study’s primary endpoints were anastomotic leak rate and anastomotic stricture rate. Secondary endpoints were set at 30- and 90-day mortality rates. All outcomes including postoperative complications (Clavien-Dindo >III) were recorded prospectively and analyzed retrospectively.o with arms apart and the operating surgeon standing in between the legs, with the first assistant on the patient’s left and with the second assistant on the patient’s right side. Two 5 mm port incisions  and one left flank at the anterior axillary line—for the first assistant) and three 12mm ports  were placed under direct vision . The lesser omentum was then dissected, starting from the duodenal bulb, following the hepatoduodenal ligament and then close to the liver edge cephalad towards the phreno-esophageal membrane, finishing on the right diaphragmatic crus. D2 lymphadenectomy with dissection of right gastric, left and right gastroepiploic, left gastric, celiac, splenic, and common hepatic nodes was performed as part of the standard procedure . The firThe esophagus was then circumferentially dissected free of surrounding tissue up to the lower mediastinum; the vagi nerves were identified and sectioned at the lower esophagus. An umbilical tape was placed around the gastroesophageal junction to help retract the stomach caudally and divide the abdominal esophagus. At this point, the cancer-free esophagus was transected with endoscopic linear stapler, with 60 mm purple cartridges . A 50 cm Roux jejunal loop was created and brought into the upper abdomen via a transmesocolic route  in preparation for the forthcoming esophago-jejunal anastomosis .The surgeons’ right working port-site (in the left upper quadrant) was converted to a 4–5 cm long transverse mini-laparotomy for specimen extraction. The mini-laparotomy was then sealed using the Alexis cup system .The anastomosis began by suturing of the esophageal staple line to the antimesenteric border of the jejunal loop with a 3/0 PDS suture  in an end-to-side manner, forming the posterior, outer layer of the anastomosis .Anterior wall esophagotomy using hook diathermy, 1–1.5 cm above the esophageal staple line and jejunal enterotomy of equal size was created, with subsequent approximation of the two, forming the posterior inner, full-thickness layer of the esophago-jejunal anastomosis. Two 3/0 barbed sutures  were used for this anastomotic layer, starting at the middle of the posterior layer, and running opposite to each other to reach each side of the anterior aspect of the anastomosis.At that stage and prior to completion of the anterior anastomotic inner layer, a naso-gastric tube was placed across the anastomosis under direct vision, in essence stenting the posterior anastomotic wall and allowing better visualization and suturing of the anterior anastomotic wall; the tip of the naso-gastric tube was left in the jejunal loop to facilitate an anastomotic methylene blue leak test at the end of the formation of the anastomosis.At the anastomotic corners, the same barbed sutures were then reversed to provide a forehand stitch and the anterior inner anastomotic full-thickness layer was then completed .Finally, an anterior, outer seromuscular esophago-jejunal layer using another 3/0 barbed suture  in a running fashion (Lembert sutures) was added to reinforce the anastomosis . A methySubsequently, a side-to-side mechanical jejuno-jejunal anastomosis was constructed between the alimentary and biliopancreatic limbs to complete the restoration of the gastrointestinal tract. This anastomosis was performed with the use of an endoscopic linear stapler with purple cartilages of 60 mm ; enterotomies were closed in a single layer, using 3/0 PDS sutures .The mini-laparotomy was closed using PDS loop sutures . After a final meticulous inspection of the abdominal cavity for bleeding and before releasing the carbon dioxide from the abdomen, a Robinson’s drain was placed through the left working port, across the Morrison’s space , with the drain tip lying close to the newly formed esophago-jejunal anastomosis.All patients were transferred to the ward postoperatively without a naso-gastric tube; they all remained nil by mouth for 72 h, when oral fluids were commenced if there was no clinical deviation from the norm. On postoperative day 6, all patients with uneventful recovery were on a soft diet and discharged a day later. A soft diet was continued for the next 30 days. Thereafter, return to normal diet was suggested in all patients.Statistical analysis was performed based on the curative intention of surgical procedures. All variables are reported as means and medians with their corresponding standard deviations and ranges. Statistical analysis was performed using SPSS software version 22.0 .n = 40 consecutive 3D-assisted laparoscopic total gastrectomies were performed in our department by the same surgical team, for the treatment of proximal gastric and gastroesophageal junction Siewert type III tumors. All operations were identical in terms of patient positioning, port placement, extend of lymphadenectomy and laparoscopic hand-sewn construction of the EJ anastomosis.Between January 2018 and June 2021, The male-to-female ratio was 3.4/1, with a mean age of 65 years. While neoadjuvant chemotherapy was administered to 75% of patients, 86.66 % of that cohort received the full peri-operative scheme. Radiotherapy was not administered to any of the patients. Patients’ demographics and pre-operative interventions are presented in There was no blood loss >200 mL and there were no conversions to open. The median operative time was 240 min, while the median time taken for the construction of the EJ anastomosis was 55 min. The intra-operative methylene blue leak test was negative in all cases and no anastomotic leak was noted during the postoperative period. There were no anastomotic strictures seen and the 30- and 90-day mortality rates were zero. The operative and clinical outcomes are presented in n = 35. The histopathological outcomes are presented in Most patients had poor or moderate tumor differentiation, with tumor T-status of greater than pT2 in 52.5% of the patients; positive nodal disease was present in 65% of the cases. Median number of resected lymph nodes was Laparoscopic total gastrectomy for cancer is on the rise and its utilization on upper gastrointestinal diseases has long been established, offering all the short-term advantages of minimally invasive surgery. Laparoscopic distal gastrectomy was first performed in 1992 by Goh et al. , while tIn their studies, Xiong, J.J. et al. and Shi, Y. et al. showed that laparoscopic total gastrectomy can provide favorable clinical outcomes compared to open surgery; intra-operative blood loss, overall postoperative complication rate and length of hospital stay are reduced in a statistically significant degree with the laparoscopic approach ,15. SubsThe rate-limiting step of TLTG is the construction of EJ anastomosis. In the first cases of LTG gastrectomy, esophago-jejunal anastomosis was performed with a circular stapler in an end-to-side manner, through a mini-laparotomy . NumerouIntracorporeal construction of the EJ anastomosis via endoscopic linear stapler has been the preferred method of reconstruction by most surgeons worldwide . Two appMultiple ways of constructing the anastomosis using circular staplers have been proposed, both intra-corporeally and extracorporeally . The anvOverall, linear-stapled anastomosis is considered easier to perform compared to the circular-stapled anastomosis ,33. AnotTo the best of our knowledge, there is limited data regarding laparoscopic hand-sewn EJ anastomosis during TLTG for cancer; the first report was by So et al. in 2011 , followeThis study presents our surgical technique of TLTG, utilizing hand-sewn EJ anastomosis in a two-layer fashion. Our team began to perform laparoscopic total gastrostomies for gastric and esophago-gastric junction tumors in 2013. Initially, we constructed the EJ anastomosis with a circular stapler. However, since mid-2016 and following the refinement of our laparoscopic skills coupled with the excellent results of the hand-sewn esophago-gastric anastomosis during totally minimally invasive 2-stage esophagectomy ,39, we hAll operations in our cohort were performed laparoscopically using three-dimensional (3D) optics, utilizing an Endoeye 3D Video Telescope; with 30° tip . We have been using 3D telescopes since 2016, and we have concluded that laparoscopic view was significantly improved with the glasses-based 3D optical system using the 30° tip camera in comparison to the relevant 2D optical system. Safer and easier laparoscopic construction of the EJ anastomosis as well as more precise lymphadenectomy were made feasible, mainly due to better depth perception with the 3D camera, thus potentially implying an improvement in postoperative and oncological outcomes.While laparoscopic EJ anastomosis is technically one of the most demanding ones to perform, we found it to be safe and reproducible in the hands of experienced laparoscopic surgeons. In the forty consecutive cases of our cohort, anastomotic leak rate, anastomotic stricture rate, 30-day and 90-day mortality rates were all zero.The study’s limitations are its retrospective nature coming from a single institution and due to its relatively small cohort size. Further large-scale studies and preferably randomized clinical trials are needed to elucidate this anastomotic technique and to confirm its safety, efficacy and reproducibility in laparoscopic total gastrectomy.Hand-sewn esophago-jejunal anastomosis during laparoscopic total gastrectomy for gastric and esophago-gastric junction cancer is technically demanding. However, it provides excellent results in the hands of experts, with lower anastomotic leak and stricture rates compared to other anastomotic techniques, and may lead to a paradigm shift. Further studies are needed to shed light on the short- and long-term outcomes and to supplementarily investigate the adoptability of the technique by a wider surgical audience.


Studying changes in collagen deformation behavior at the nanoscale due to variations in mineralization and hydration is important for characterizing and developing collagen-based bio-composites. Recent studies also find that carbon nanotubes (CNTs) show promise as a reinforcing material for collagenous bio-composites. Currently, the effects of variation in mineral, water, and CNT content on collagen gap and overlap region mechanics during compression is unexplored.We use molecular dynamics simulations to investigate how variations in mineral, water, and CNT contents of collagen bio-composites in compression change their deformation behavior and thermal properties. Results indicate that variations in mineral and water content affect the collagen structure due to expansion or contraction of the gap and overlap regions. The deformation mechanisms of the gap and overlap regions also change. The presence of CNTs in non-mineralized collagen reduces the deformation of the gap region and increases the bio-composite elastic modulus to ranges comparable to mineralized collagen. The collagen/CNT bio-composites are also determined to have a higher specific heat than the studied mineralized collagen bio-composites, making them more likely to be resistant to thermal damage that could occur during implantation or functional use of a collagen collagen/CNT bio-composite biomaterial. Experiments by Holmes et al. find that telopeptide cross-link cleavage alters the flow of heat and structure of collagen  are being studied as a material to strengthen collagen-based bio-composites . In this study, MD simulations are performed using the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) program Plimpton . DetailsDetails of the development of the CNT models for the collagen/CNT bio-composites are available in the S2 and S3 sections of the supplementary material. We vary the water content from 10 wt% to 20 wt% for a bio-composite with 10 wt% single-walled carbon nanotubes (SWCNTs). Higher water contents of 10–20 wt% are chosen for the collagen/CNT composites in order to compare their mechanical properties to the non-mineralized collagen composites with the water content of healthy fibrils (10–20 wt%), while the water contents of the mineralized composites were varied until they reached modulus values comparable to the collagen/CNT composites with 10–20 wt% water content. We also compare collagenous bio-composite with 7 wt% SWCNTs with 11 wt% multi-walled carbon nanotubes (MWCNTs). x-direction reaches the target stress value. The compressive stress is plotted vs. the linear strain, the Young’s modulus is plotted vs. the water content, and we plot the gap/overlap ratio vs. applied stress. The gap and overlap region lengths are determined by the measuring distance between the terminal amino acid residues of the collagen molecules, which corresponds to the beginning and end of the gap and overlap regions. This can be seen in the schematic of The method for applying quasi-static stress is the same as used in a previous study  agrees well with the experimental results from other studies in 3.3Next, we analyze the gap/overlap ratio shown in  and atomIn D-period of the simulated bio-composites. This agrees with the results of the previously mentioned experiment  to 10 w−1K−1 (Pradhan et al. We compare the specific heat capacity for each of our models to the specific heat capacity values determined by Qu and Tomar (Qu and Tomar 4.We developed models of collagenous bio-composites with varying mineral, water, and CNT content and tested them under compressive loading using molecular dynamics to determine the viability of CNTs as a substitute for mineral in collagenous bio-composites. In addition, we studied the deformation behavior of the gap and overlap regions of the bio-composites and calculated their heat capacities. An increase in the mineral content increased the stiffness of the bio-composites and reduced the change in elastic modulus caused by the inclusion of water. The inclusion of CNTs in non-mineralized bio-composites also increases the elastic modulus of the bio-composites to ranges comparable to the modulus values of mineralized bio-composites. There is evidence of the shearing of collagen molecules between mineral molecules in the gap region, but at lower mineral contents the bending of collagen molecules is also observed. These observations were used to formulate a model to determine the modulus of mineralized bio-composites. The formulated model agrees with the simulations of this study, as well as modulus values determined in experimental studies by others.For non-mineralized bio-composites at low water contents, the water occupies gap region voids, causing gap region expansion and overlap region contraction. At higher water contents some water also occupies overlap region voids, which reduces the overlap region contraction, and when stress increases, the gap region deforms more than the overlap region. In mineralized bio-composites, the water primarily occupies overlap region voids because of mineral in the gap region, which causes the overlap region to expand while the gap region contracts. As the stress increases, the gap/overlap ratio remains approximately constant. CNTs in non-mineralized bio-composites occupy gap region voids, with the amount and distribution of CNTs and water determining the relative deformation between gap and overlap regions.The collagen/CNT bio-composites have a much higher heat capacity than the mineralized bio-composites and a lower total bio-composite mass. Overall, collagen/CNT bio-composites have stress vs. strain behaviors, elastic moduli, and gap/overlap ratios comparable to those of mineralized bio-composites, but with a higher heat capacity that would allow collagen/CNT bio-composites to be less susceptible to thermal damage during implantation or functional use in applications like bone machining, as pointed out by Qu and Tomar (Qu and Tomar