Itga4  epicardial deletion in mice and cryocauterisation of chick embryonic hearts disrupted epicardial development and ventricular wall growth, two essential events in coronary embryogenesis. Our results suggest that myocardial discontinuities in the embryonic ventricular wall promote the early contact of the endocardium with epicardial-derived coronary progenitors at the cardiac surface, leading to ventricular endocardial extrusion, precocious differentiation of coronary smooth muscle cells, and the formation of pouch-like aberrant coronary-like structures in direct connection with the ventricular lumen. The structure of these CAF-like anomalies was compared with histopathological data from a human CAF. Our results provide relevant information for the early diagnosis of these congenital anomalies and the molecular mechanisms that regulate their embryogenesis.Coronary Artery Fistulae (CAFs) are cardiac congenital anomalies consisting of an abnormal communication of a coronary artery with either a cardiac chamber or another cardiac vessel. In humans, these congenital anomalies can lead to complications such as myocardial hypertrophy, endocarditis, heart dilatation, and failure. Unfortunately, despite their clinical relevance, the aetiology of CAFs remains unknown. In this work, we have used two different species (mouse and avian embryos) to experimentally model CAFs morphogenesis. Both conditional  Insights from animal models highlight specific disturbances in early heart development as a likely point of origin for the formation of coronary artery fistulas (CAF), abnormal linkages between one of the coronary arteries and other regions of the heart. Children born with such defects are at heightened risk of serious heart problems. Using chick and mouse embryos, José Pérez-Pomares at the University of Málaga in Spain revealed a mechanism by which CAFs may arise in humans. Early in development, the heart muscle that forms the ventricle chambers undergoes a process of growth and compaction that transforms ventricular walls into thick and dense tissue layers. The researchers demonstrated that genetic or physical disruptions that interfere with this process give rise to CAF-like features, highlighting a promising strategy for modelling this congenital defect. Despite the remarkable social impact of CHD, not much it is known about the embryological defects underlying the origin of many of these conditions. This lack of information on the cellular and molecular mechanisms that cause CHD limits our chances of discovering intrinsic and extrinsic factors that potentially disrupt normal embryonic heart development, and novel early diagnostic markers for these ailments3. We are thus still far from having enough information to design and implement an effective CHD prevention strategy. In this work, we provide experimental, mechanistic evidence on the origin of a distinct form of CHD that affects coronary blood vessels and other cardiac structures.Congenital heart disease (CHD) affects around 1% of new-borns4. Some CA anomalies can severely disrupt cardiac function and even result in sudden cardiac death. It has been reported that 0.6–1.5% of patients undergoing invasive cardiovascular imaging have coronary anomalies, including coronary artery fistulae (CAFs)6. CAFs are abnormal communications between a coronary artery and other cardiovascular cavity that often are asymptomatic, but can also cause long-term variations in the systemic hemodynamics, normally due to the arrest of part of the blood circulation that may result in ischaemia. The two main sub-types of CAFs described in the literature involve the connection of a coronary artery to either the lumen of a cardiac chamber  or a large cardiac vessel (coronary arterio-venous fistula). The most common CAF type reported in the literature is the arterio-ventricular one (41%), followed by coronary artery connections to the right atrium (26%), the pulmonary trunk (17%), the coronary sinus (7%), the left atrium (5%), and the left ventricle (less than 3%)7. In particular, arterio-ventricular fistulae involving large coronary vessels often manifest as a coronary wall convexity (dilation) forming a conspicuous cameral, pouch-like structure at the cardiac surface. Although studies differ in their conclusions on CAF incidence in the right versus the left ventricle8, solitary ventricular CAFs were found to be more frequent than multiple microfistulas9.Coronary artery (CA) anomalies are a well-characterized subset of CHD10. Importantly, these cardiac congenital defects should not be mistaken for isolated connections existing between blood vessels and /or cardiac cavities that result from surgical interventions, traumatic events, or infections , which are often referred-to as ‘fistulae’ and even ‘pseudoaneurisms’13. Indeed, congenital CAFs are true anomalies that derive from abnormal embryogenesis and, as such, they frequently associate to other forms of CHD, including Tetralogy of Fallot, atrial and ventricular septal defects (VSDs), persistent ductus arteriosus, pulmonary atresia and left ventricular non-compaction, among others8. Some authors have attempted to relate CAFs with gross genetic abnormalities such as chromosome deletions, e.g., 22q11.214 and conditions involving chromosome number variation like Turner, Klinefelter, or Down syndromes15, but so far the pathogenesis of CAFs remains largely unknown.It is well established that the vast majority of CAFs identified at birth have a congenital originseptum transversum/proepicardium (ST/PE)18. This finding implies that, in order to complete the coronary vascular circuitry, cells from the inner ventricular endocardium  and the outer epicardium , merge by crossing the cardiac chamber myocardium that separates them. Moreover, the epicardium, whose contribution to the forming coronary vasculature  is well-known21, also acts as an instructive signalling centre that promotes the growth and the maturation of the adjacent myocardium22. Therefore, it is very likely that both anomalies in the cells that build the coronary vascular tree and the disruption of myocardium growth and maturation are involved in the origin of CA anomalies. In accordance with this hypothesis, animal models for defective epicardial development display a thin compact ventricular myocardium23 and coronary defects25, some of which are reminiscent of CAFs26.Approaching the mechanistic origin of CAFs requires a detailed understanding of embryonic coronary vascular and ventricular wall formation and maturation. Recent studies have demonstrated that coronary arteries form from a primary endothelial capillary network, including cells from the endocardium (sinus venosus and ventricle), and the epicardial progenitors at the Itga4 (α4-integrin) gene in epicardial progenitors. Itga4 encodes for the α4 integrin subunit, whose cardiac expression is restricted to the epicardium. Integrin dimers including α4 integrin subunit promote epicardial adhesion to the myocardium by interacting with the myocardial-expressed Vcam-1 ligand. Since mice with systemic Itga4 loss-of-function show an extreme phenotype and die around embryonic day (E) 9.5–1027, we have used the Cre/LoxP system to circumvent this early embryonic lethality while preserving the anomalous epicardial formation and the myocardial defects that are secondary to epicardial developmental anomalies. In avian embryos, local loss of myocardial integrity is directly achieved by cryocauterisation of the ventricular wall without the involvement of primary epicardial defects26. To uncover the cellular and molecular mechanisms underlying CAF pathogenesis, transcriptomic analysis of mutant mice and careful immunohistochemical characterization of Itga4 mutants and cryocauterised avian embryonic hearts have been carried out. Finally, results from these experimental studies were compared with histopathological data from a human paediatric CAF to confirm our experimental findings.In this work, we hypothesize that the disruption of embryonic ventricular compact myocardial growth leads to the formation of CAF in both mammals and avians. Compact myocardium growth arrest in mouse embryos is achieved indirectly, i.e., by disrupting epicardial formation and the subsequent depletion of the epicardial-derived pro-mitotic factors that regulate cardiomyocyte proliferation. To interfere with murine epicardial development, we have conditionally deleted the guidelines for animal care and welfare.All animals used in our research program were handled in compliance with the European and Spanish Itga4 systemic ablation28, this gene was conditionally deleted in the ST/PE by using a G2-Gata4-Cre mouse transgenic line18. Itga4flox and G2-Gata4-Cre mice have been previously described30. To track epicardial derivatives, G2-Gata4-Cre mice were also crossed with Rosa26R-YFP mice18. Heterozygous G2-Gata4Cre/+ were bred with Itga4flox/+ mice to generate G2-Gata4Cre/+;Itga4flox/flox mutants. .To circumvent the early lethality of E9.5 embryos were fixed, embedded in paraffin, and sectioned in sterile conditions. In situ hybridization was performed following standard protocols (see supplementary information). The probe was generated with the forward 5′-ATGGTAACCGTAGCTGTACCT-3′ primer and the reverse 5′-AGTCATCCTTGTTCCCACTTG-3’ primer.For routine histological analyses, embryos or embryonic hearts were fixed, embedded in paraffin, and sectioned. Tissue sections were dewaxed in xylene, hydrated in ethanol series, and rinsed in distilled water. Samples were then submitted to Mallory’s trichrome staining (see supplementary information).For immunohistological analyses, embryos were fixed in a 4:1 methanol:DMSO solution overnight at −20 °C, dehydrated in an ethanolic series, embedded in paraffin, and sectioned in a microtome. Immunofluorescence of the tissue was performed following standard methods (see Supplementary Information). Primary and secondary antibodies used in this study are listed in Supplementary Tables A working volume of 420–450 µl from an aqueous BrdU  stock solution (10 mg/mL) was injected intraperitoneally in pregnant females 30 min before embryo extraction. Embryos were fixed in a 4:1 methanol:DMSO solution overnight, embedded in paraffin, and sectioned with a microtome. Sections were dewaxed, rehydrated, treated with 2 N HCl (30′ at room temperature), and washed again in 100 mM sodium tetraborate (5′). Following steps and image acquisition were performed according to standard protocols (see supplementary information). Proliferating cardiomyocytes were estimated with the IMARIS® software (see supplementary information). A t-test and a post hoc Lubischew coefficient analyses were used to assess statistical significance.G2-Gata4+/+;Itga4flox/+ control and G2-Gata4Cre/+;Itga4flox/flox mutant E11.5 embryos using the Arcturus Picopure RNA isolation kit (Applied Biosystems), including a step for removing genomic DNA. For each genotype, RNA coming from 3 ventricles was pooled (n = 4–5). Quality control was performed on an Agilent 2100 bioanalyser using an RNA 6000 Pico LabChip kit (Agilent Technologies). The RNA integrity number (RIN) was 10 for all the samples. cDNA was prepared using the standard Illumina TrueSeq RNASeq library preparation kit. Libraries were sequenced in a GAIIx Illumina sequencer using a 75 bp single end elongation protocol. Sequencing read quality was assessed with FastQC . Contaminating Illumina adapters were trimmed with Cutadapt 1.7.1 which also discarded reads that were shorter than 30 bp. The resulting reads were mapped against the mouse transcriptome  and quantified using RSEM v1.2.20.RNA was isolated from heart ventricles of Prior to statistical analysis, genes with sufficiently large counts were identified and retained with the function filterByExpr, implemented in the R package edgeR. Data normalization and statistical testing was performed with DESeq2. Enrichment analyses for signalling pathways and gene ontology categories were performed with the R package clusterProfiler. KEGG was selected as the database for pathways and we focused on biological process ontology.Pgk1 and Ppia were selected as reference genes as previously described31. Primer sequences (Supplementary Table P < 0.05).RNA isolation and qPCR analyses were performed following routine protocols (see Supplementary Information). The melting curve analysis  and size fractionation by agarose gel electrophoresis were used to confirm amplification of the expected products. Primers used are listed in Supplementary Table VCAM1 and the second from ITGA4, representing two different cellular types, cardiomyocytes, and epicardial cells, respectively. Interactions between proteins were downloaded from StringDB and visualized in Cytoscape. Nodes were selected based on first and second PPI associations with VCAM1 and ITGA4 using a maximum of 10 connections derived from “experiments” or “databases”.A PPI sub-network was identified by selecting genes derived from the RNA-seq data analysis, then identifying the predicted proteins associated with these genes and selecting genes and interactions within the first two levels of interaction (A-B-C), considering up to 10 contacts per level (see Supplementary information for further details). As a result, two different sub-networks were identified, one from 32 and the classification for quail developmental stages33, respectively. Eggs were kept in a rocking incubator at 38 °C for 55–60 h (early HH17). A N2-cooled copper probe was used to burn the myocardial wall as previously described34, In every single case, the embryos were inspected to ensure that epicardial cell progenitors  had not yet attached to the myocardial surface. Routine histological analyses were performed as previously explained for mouse embryos .The second experimental model used in this work was the cryocauterisation of the avian embryonic heart. Chick and quail embryos were staged according to the Hamburger and Hamilton stages of chick development® software.To acquire 3D images, chick hearts were washed in PBS and fixed in paraformaldehyde for several weeks at 4 °C. Then, hearts were washed in methanol series, embedded in JB4 resin, sectioned and analyzed by a high resolution episcopic microscope (HREM). Two different stages (4 days post-injury “dpi” and 16 dpi) were analyzed. The 3D shape of the tissue was reconstructed using the ImageJChick and quail embryos were fixed in a 4:1 methanol:DMSO solution overnight, dehydrated in an ethanolic series, embedded in paraffin and sectioned in a microtome. Immunostaining of avian tissues was performed following the same protocol described in the mouse (see supplementary information). For QCPN immunostaining, epitopes were unmasked using TEG buffer  in a pressure cooker for 10 min, and the signal was amplified with TSA biotin system kit .Sharp tungsten needles, small iridectomy forceps, and scissors were used to isolate quail tubular hearts (HH 16–17) under a dissecting scope. Isolated ventricles and proepicardia were cultured in different conditions (see Supplementary Data). All samples were fixed overnight in Methanol-DMSO (4:1) solution. Fixed tissue constructs were dehydrated in an ethanol series, washed in butanol, embedded in paraffin, and sectioned in a microtome. Some aggregates were not embedded in paraffin and processed as whole mount samples.For immunohistochemistry, sections were stained following the same protocol than previously described for mouse samples (see Supplementary Information).The autopsy of a six weeks-old patient with a diagnosis of complex congenital heart disease, who had previously undergone a palliative procedure  and had died of sudden cardiac arrest, was performed at the Necker Hospital . Adequate informed consent has been obtained for the study of paediatric tissue samples. Post-mortem analysis revealed massive cardiac tamponade as a probable cause of death. Formalin-fixed samples were embedded in paraffin, sectioned with a microtome, and mounted on slides. Tissue sections were dewaxed with xylene and hydrated in an ethanolic series. Routine histological analyses were performed by Mallory’s trichrome staining as detailed above. Immunochemical analyses of the human tissue were performed as described in the supplementary information.G2-Gata4-Cre epicardial driver mouse line, displaying a strong Cre expression in ED 9.5 epicardial progenitor  cells . Crossing these two transgenic lines resulted in the conditional deletion of the Itga4 gene in epicardial progenitors , as shown by the reduced Itga4 mRNA levels found in G2-Itga4 mutant proepicardia  and the transcription factor Tcf21 was sharply reduced in mutant ventricles as compared with control ones  and mutant (G2-Itga4) embryos  were identified after comparing mutant versus control samples. Out of those 376 genes, 134 were overexpressed and 242 underexpressed in the mutant tissues , which are annotated to Gene Ontology (GO) Enrichment Analysis categories such as ‘Extracellular structure organization’ or ‘cell surface receptor signalling pathway’ are significantly reduced in the mutants. These and other genes also annotate to Pathway (KEGG) Enrichment Analysis terms such as ‘Extracellular structure organization’ and ‘Extracellular matrix organization’, which were equally downregulated in mutant ventricles , ‘Immune Effector Process´ , ´Blood coagulation’  or ‘Wound healing’  were upregulated. The pathway enrichment analysis also revealed that ‘Focal adhesion’ and ‘ECM-receptor interaction’ were the most significantly enriched pathways in control animals . The analysis identified two sub-networks of protein interactions (Ezrin(Ezr)/Radixin/Moesin(Msn) and Ncf/Cybb/Rac1) that could be altered in G2-Itga4 embryos  decrease in cycling cardiomyocytes of the compact ventricular layer  with outlet VSD, and a large coronary arterio-ventricular fistula with a pouch-like appearance Fig. . This CAG2-Itga4 mouse mutant embryos died shortly after endocardial vesicles form on the heart surface   in the developing heart Fig. . Three d+) Fig. .19  quail transplantations into chick hosts and subsequent cryocauterisation of the developing chimeric embryo revealed that the majority of smooth muscle cells (αSMA+) forming the CAF-like wall were donor-derived (quail) cells expressing the quail-specific QCPN nuclear marker  growth and accumulation at the surface of the cardiac explant  proepicardial-derived cells in close association with quail endocardial (QH1+) cells. αSMA marker expression was scarce in areas where chick cells were in contact with myocardial (αSMA+/QH1−) but not endocardial (αSMA-/QH1+) cells  and quail myocardial-endocardial tissues were developed. Hanging drop culture of quail embryonic hearts Fig.  allowed ant Fig. . Chimeriant Fig.  to theseant Fig. . After ills Fig. .6, have been known for more than a century. These anomalies are rare, but often associate to multiple cardiac complications such as myocardial hypertrophy, endocarditis, heart dilatation, and cardiac failure8.Aberrant connections between coronary arteries and a heart chamber or a large vessel, commonly referred-to as coronary arterio-ventricular fistulae (CAFs)36. This requires a primary distinction between anomalies of the coronary wall with an acquired origin, which may result from defective surgical procedures37, and congenital CAF anomalies with a developmental origin7. A note on the nature of CAF should also be made as based on strict histopathological criteria, as true congenital vascular aneurysms involve the local bulging of the three tissue layers of a vessel , whereas pseudoaneurysms  are anomalous structures with a characteristic pouch-like appearance containing tissue from the medial and adventitial mural layers only38. In our work, we have revisited the histoarchitecture of human CAF through the histological analysis of a conspicuous CAF found in a six-week-old child. This examination indicates that endothelial , smooth muscle , and fibroblastic  tissues form the wall of these structures. Detailed reports indicate that the wall in human CAFs contains smooth (but not striated) muscle bundles frequently separated by several internal elastic laminae, as well as a characteristic intimal layer showing non-specific fibrous thickening39. Similar histological features were found in the paediatric case reported in our study: the internal surface of the CAF is lined by a vascular endothelium displaying a characteristically poor Von Willebrand immunoreactivity associated to the endocardium40, with the majority of the fistula wall formed by smooth muscle cells (α-SMA+). On a final note, our analysis of this human case revealed that the ventricular myocardium was poorly compacted. This finding developmentally associates poor ventricular thickening or compaction41 with the occurrence of congenital CAF as described in paediatric and adult human patients42.A certain degree of confusion exists in the literature about the origin of CAFs. This is, at least in part, due to semantics, since the terms coronary ‘aneurysm’ and ‘pseudoaneurysm’ are often interchangeably used to refer to CAF43, no data on the disrupted developmental mechanisms that lead to CAF formation are available in the literature. Although the anatomical description of CAF may suggest these anomalies are secondary to disrupted coronary vasculature morphogenesis44, it has also been suggested that they could form by the persistence of sinusoidal connections between the forming coronary arteries and the primitive tubular heart lumen45. These two hypotheses, reinterpreted in light of recent discoveries on the embryonic origin of coronary blood vessels18, are fully compatible and could explain the origin of part of congenital CAF. Indeed, connections between both the embryonic sinus venosus at the venous pole of the heart and the ventricular lumen with the primitive coronary capillary plexus occurs during normal coronary embryonic development18. Such connections are progressively lost as the ventricles thicken and mature during the perinatal stages46. Thus, the proper growth of ventricular myocardium is necessary to maintain the patterning and stabilization of embryonic coronary vessels and is also required to separate coronary endothelium from its endocardial origin. In this work, we suggest that the anomalous disruption of compact myocardial wall formation may result in persistent communications between coronary blood vessels and the cardiac lumen leading to CAF formation.While the congenital origin of CAFs is widely acceptedItga4 gene. This conditional mutant recapitulates the severe ventricular compact myocardial layer defects, known to be secondary to the anomalous development of the epicardium, found in Itga4 systemic mutants27, but survive beyond embryonic day 9.5. Relevant to our approach, Itga4 mutants represent an extreme case of cell (Itga4) and non-cell autonomous (Vcam1) defects associated to defective epicardial development, a key developmental process required for both coronary blood vessels and ventricular wall formation23.To test our working hypothesis, we used two animal models. The first one is a mouse with an epicardial deletion of the in ovo experimental manipulations with little or no impact on embryo viability47. In particular, we have taken advantage of a recently described procedure allowing for the creation of restricted damage on embryonic tissues by means of local cryocauterisation34. In this case, cryocauterisation was used to locally disrupt ventricular myocardial wall continuity prior to the formation of the epicardium. In this experimental setting, the epicardium remains unaffected, allowing for the study of epicardial mesenchymal derivatives in the damage area where CAF will form.Our second animal model is the chicken embryo, which is amenable to a large variety of Itga4 gene in mouse epicardial progenitors and epicardial cells disrupts early epicardial development, significantly reducing the number of epicardial cells as shown by histological and epicardial-specific gene expression analyses. Abnormal epicardial development in these mutants is concomitant with the lack of thickening of the ventricular compact myocardial layer due to reduced cardiomyocyte proliferation, as it has been described for other epicardial mutants48. Such discontinuities allow for the extrusion of the endocardium, which can be distinguished from the forming coronary vascular endothelium by its strong endomucin expression. Indeed, endomucin marker expression confirms the endocardial origin of the tissue lining the CAF cavity, also suggesting it does not derive from the differentiating coronary vascular endothelium found in the cardiac surface (endomucin-negative).Genetic deletion of the Itga4 mutants revealed a significant increase in genes associated to tissue damage  as well as a marked decrease in genes linked to cell surface signalling and ECM organization. These findings, taken together, strongly suggest that epicardial-to-myocardial adhesion mechanisms, including those mediated by the ECM, were affected by Itga4 deletion. Consequently, we checked the status of Vcam-1, the canonical α-4 integrin epicardial ligand49. Interestingly, Vcam-1 gene expression level was unchanged in Itga4 epicardial mutants, but Vcam1 protein distribution in the plasma membrane of cardiomyocytes was found to be severely altered in these animals. Both the reduced number of epicardial differentiating cells and the altered secretion of subepicardial matrix proteins in mutant embryos could explain the poor cell surface organization of Vcam1. Our in silico protein interaction modelling suggested that different proteins belonging both the Ezrin/Radixin/Moesin and Ncf/Cybb/Rac1 pathways could potentially interact with Vcam1. It is tempting to think these pathways could be involved in the developmental origin of the mutant phenotype, so that disruption of ventricular cardiomyocyte polarity may have an impact on ventricular cardiomyocyte cohesiveness and this, in turn, result in myocardial discontinuities. This hypothesis, however, needs a further, detailed analysis, as chamber cardiomyocyte polarization is far from been complete at the earliest embryonic stages studied in our work (E9.5). The specific cellular context of genes related to Itga4 is suggested by the high prevalence of epithelial  specific proteins in the model. Moreover, our model also suggests the existence of a biochemical/functional relationship between Itga3 and Itga4. Whether anomalies in Itga4-Itga3 protein interactions could promote the previously described CAF-like phenotype needs further experimental analysis. Taken together, these results indicate that defective cell-to-cell and cell-to-matrix adhesion could underlie the appearance of the characteristic ventricular myocardial discontinuities of epicardial Itga4 mutants. In turn, these ventricular myocardial wall discontinuities could represent a passage through the compact myocardium for the endocardium, providing a physical continuum between the endocardial lining of the cardiac chamber with the pericardial space.The transcriptomic analysis of epicardial Itga4 epicardial mouse mutants at E12.5–13 prevented us from studying the formation of the CAF-like structures in these animals in more detail. To progress in our analysis of these anomalies, we decided to use chick embryos, a highly manipulable experimental animal model extensively used to study embryogenesis. Hence, we carried out limited cryocauterisation of the chick embryonic heart  to reproduce the ventricular myocardial discontinuities we identified in Itga4 epicardial mutants. Our results show that the mechanical disruption of the chick embryonic myocardium faithfully reproduces the formation of arterio-ventricular anomalies showing a structure very similar to that described for human CAF. The combination of cryocauterisation and interspecific quail proepicardium-to-chick chimerisation for cell fate tracking demonstrates that the smooth muscle wall covering these anomalies, as that of coronary arteries, has epicardial origin23. Our results also confirm that the inner lining of the fistula cavity is not an epicardial derivative; the absence of the QCPN quail donor marker and the continuity of these cells with the ventricular endocardium strongly suggest a host endocardial origin for this tissue. The vascular lining of the fistula, which is always continuous with the endocardial ventricular lumen, can appear connected with the endothelium of close coronary vessels, although this feature is not frequent in our samples. This may be explained by the early formation of the pouch-like structure, that seems to derive from the ventricular endocardium rather than from the coronary endothelium. Therefore, formation and muscularisation of the CAF seems to precede coronary blood vessel formation, precluding early connection between the pouch-like lumen and coronary vascular endothelium. Since coronary smooth muscle is known to be a major embryonic epicardium derivative23, and endothelial-to-mesodermal progenitor cell contact and paracrine molecular cross-talk efficiently result in smooth muscle differentiation50, we tested in vitro whether early endocardium-to-epicardial-derived cell contact could effectively induce smooth muscle differentiation from the latter cell type. Our results convincingly demonstrate that early in vitro endocardium-to-epicardial-derived mesenchymal cell interaction triggers epicardial cells differentiation into smooth muscle cells, as normally happens in vivo.The death of We have herein shown that myocardial discontinuities in the embryonic ventricular wall, either secondary to anomalous epicardial development or resulting from a direct damage to the embryonic myocardium, cause CAF in two different vertebrate models via the extrusion of the endocardial lining of the developing heart, the precocious and anomalous formation of a smooth muscle wall over the extruded endocardium, and the persistence of arterial to ventricular lumen connection Fig. . The hisSupplementary material


Cocaine use disorder (CUD) is an intractable syndrome, and rising overdose death rates represent a substantial public health crisis that exacts tremendous personal and financial costs on patients and society. Sharp increases in cocaine use drive the urgent need for better mechanistic insight into this chronic relapsing brain disorder that currently lacks effective treatment options. To investigate the transcriptomic changes involved, we conducted RNA sequencing on two striatal brain regions that are heavily implicated in CUD, the nucleus accumbens and caudate nucleus, from men suffering from CUD and matched controls. Weighted gene coexpression analyses identified CUD-specific gene networks enriched in ionotropic receptors and linked to lowered neuroinflammation, contrasting the proinflammatory responses found in opioid use disorder. Integration of comprehensive transcriptomic datasets from mouse cocaine self-administration models revealed evolutionarily conserved gene networks in CUD that implicate especially D1 medium spiny neurons as drivers of cocaine-induced plasticity. Cocaine use disorder alters evolutionarily conserved striatal gene networks of neuroinflammation and ionotropic receptors. Cocaine use disorder (CUD) is a major public health problem that is associated with substantial morbidity and mortality. Within the United States, the rate of cocaine overdose deaths has markedly risen—almost threefold—over the past decade  on both the ventral (NAc) and dorsal (CN) striatum from postmortem tissue of persons with CUD and matched control subjects. Our study uses the largest and most diverse cohort examined to date. We combined differential gene expression and network-based approaches to provide an integrative and unbiased characterization of transcriptional signatures triggered by chronic cocaine use across the striatum. Our analyses revealed a profound overlap between CUD-specific transcriptome changes in the NAc and CN, introducing the down-regulation of neuroinflammatory processes and up-regulation of synaptic transmembrane transporters and ionotropic receptors.Using weighted coexpression analyses, we identified CUD-specific gene networks as likely drivers of the transcriptomic alterations observed in CUD, which prominently implicate synaptic remodeling downstream of cyclic adenosine monophosphate (cAMP) signaling. Because of the inherent constraints of developing a mechanistic understanding of CUD-related transcriptional activities in human striatum, it is essential to understand underlying molecular mechanisms in animal models of cocaine use. However, a key unanswered question is whether these preclinical models, in fact, reflect disease mechanisms seen in human CUD. We thus investigated whether recent preclinical findings of transcriptomic alterations in mouse models of cocaine self-administration (SA) can, in fact, recapitulate the transcriptome changes that we uncovered in persons with CUD. Our multiple-level analysis revealed robust converging pathways and evolutionarily conserved gene networks in mice and humans that characterize striatal transcriptional and physiological mechanisms in CUD.Notably, the striatum is composed of two types of GABAergic projection neurons, medium spiny neurons (MSNs), which together constitute ~95% of the total neural population in this brain area (N = 25) compared to well-matched control subjects (N = 20). Our study was balanced to include both non-Hispanic white and Black individuals (table S1), who have been shown recently to be disproportionately affected by cocaine-related overdose deaths  threshold of 0.0001; P = 2.8 × 10−5 and D1 MSNs P = 6.7 × 10−4, Fisher’s exact, pSI threshold of 0.01; In contrast, PCA identified the feature brain region as the primary source of sample variance. Thus, we subsequently included brain region as a covariate in the design of all differential gene expression analyses using DEseq2 (fig. S1A). We first confirmed that both NAc and CN transcriptomes were, in fact, representative of the adult human striatum and no other brain regions by using brain region–specific expression analysis (SEA) that was built on human data from the Brainspan collection  ± 0.26; false discovery rate (FDR) ⩽ 0.1 adjusted differentially expressed transcripts (DETs) indicated for NAc (25) and CN (7); P = 3.7 × 10−2; We next investigated the impact of CUD on gene regulation in the NAc and CN. Differential expression volcano plots showed an even contribution of down-regulated (693) and up-regulated (728) transcripts in CUD in the NAc with more contribution of up-regulated transcripts in the CN [down: 640, up: 949; statistical threshold Using two-sided rank-rank hypergeometric overlay (RRHO) to compare patterns and strength of transcriptional overlap in a threshold-free manner . HeatmapTo gain insight into the enrichment of DETs and their molecular functions in the two brain regions, we performed Gene Ontology (GO) analysis (ShinyGO v0.75). Given the nonsignificant overlap of DETs between the two regions, the NAc and CN were analyzed separately. Molecular function GO terms for the up-regulated DETs in the NAc were associated with transmembrane transporter activity, particularly, glutamate transporters, while down-regulated DETs were enriched in signal receptor binding as it relates to inflammatory responses, interleukin (IL) signaling, and oxidative processes , and a fP ⩽ 0.05, FDR = 10%, magnitude threshold logFC ± 0.26). There were no differences in the biotypes making up DETs between the control and CUD groups (fig. S3C). Furthermore, there was significant overlap (2047 DETs) between control and CUD subjects, and a heatmap of overlapping DETs showed highly similar patterns of expression in these transcripts, regulating voltage-gated ion channel activity .To further examine the effects of CUD on transcriptomic differences between the NAc and CN itself, we performed differential expression analysis contrasting the NAc and CN transcriptomes in both control and CUD individuals . CUD is linked to an increase in differential gene regulation between these two brain regions, with a 10-fold overall expansion in the number of DETs compared to control subjects  located near CUD-related DETs were enriched for genetic risk variants linked to substance use–related traits and psychiatric disorders using partitioned linkage disequilibrium score regression . Risk taWhile animal models have been a major focus of research to better understand the molecular pathophysiology of addiction, it remains uncertain whether the cocaine-related molecular alterations explored in rodent models mirror transcriptomic changes in humans with CUD. To assess the ability of cocaine self-administration in mice to mirror transcriptional changes in individuals with CUD, we conducted a direct comparison of the human CUD transcriptomic patterns and those in a mouse cocaine self-administration study where transcriptional regulation was assessed in the NAc using RNA-seq  (17). InThe finding that the human transcriptional abnormalities most closely resembled those seen in mice shortly after chronic cocaine self-administration is consistent with the fact that most individuals included in our cohort were active drug users at the time of death. By comparing the transcriptional landscapes of self-administering mice and humans with CUD, these data establish the ability of mouse models to reflect disease mechanisms and provide strong support for the validity of reverse and forward translation approaches.IL-6, IL-1β, and TNFα , which generates transcriptional networks based on the correlated expression of genes across a population of samples . One majP < 0.05; table S5), of which five networks were characterized by high differential connectivity in CUD , a network that showed enrichment for DETs in the human NAc and differential tissue correlation of its CUD-related gene responses , synaptogenesis signaling , and dopamine-DARPP32 feedback in cAMP signaling . DARPP32 is a well-known phosphoprotein in dopaminoceptive neurons that modulates electrophysiological, transcriptional, and behavioral responses to cocaine and other drugs of abuse , the striatal receptor for prostaglandin E2 that is produced in response to D1 and D2 dopamine receptor stimulation .We again wanted to leverage the integration of human subjects with CUD to mouse self-administration models. Thus, we investigated whether the human CUD-specific modules are conserved in the widely used mouse model of cocaine self-administration. We conducted a direct comparison of the CUD-specific human gene networks and performed WGCNA on a recently published comprehensive NAc transcriptome data from the mouse cocaine self-administration mentioned above  and glutamate receptor subunits  that have been previously implicated in the behavioral manifestation of addiction , which have also been shown to be regulated in the NAc of rhesus macaques with long-term cocaine self-administration (NR4A1 (green), NR4A2 (thistle), and NR4A3 (crimson). These transcription factors have been identified as critical for CREB-mediated gene responses in rodent models of cocaine self-administration and are hypothesized to interact with CREB to regulate different transcriptional programs in a context-specific manner .To investigate and further characterize the molecular processes controlled by these conserved networks, we performed GO enrichment analyses on both the human skyblue and crimson modules, as well as on the corresponding mouse modules . The human skyblue network was enriched in components of synaptic membranes that regulate the activity of ionotropic receptors and transmembrane transporters , among tddiction  also incstration , implicaDrd1a::Egfp-L10a or Drd2a::Egfp-L10a) that enabled fluorescent-activated nuclei sorting for either cell type combined with population-specific RNA-seq. The following groups were included in this mouse study: effects of acute cocaine (1 hour) in drug-naïve mice, effects of prolonged withdrawal (30 days) from chronic cocaine, and effects of an acute cocaine challenge (1 hour) after prolonged withdrawal from chronic cocaine, all compared to saline-treated control animals.To gain insight into the potential involvement of D1 and D2 MSNs in the CUD-specific gene modules that were conserved in cocaine self-administering mice see , we usedIl-6 is one of the significantly down-regulated genes in the NAc of subjects with CUD, indicating that D1 MSNs partially drive the transcriptional response within this module.We first evaluated cell type–specific transcriptional responses in the conserved human crimson module  using κ-In the conserved human lightyellow module, most of genes were primarily induced in D1 MSNs—but not in D2 MSNs—by acute cocaine and cocaine relapse . This coThe results of our study map transcriptome-wide changes in gene expression in two striatal regions of humans with CUD. We identify cocaine-related gene regulation as well as highly regulated gene networks in NAc and CN that are most prominently affected by this syndrome. While these two brain regions exhibit distinct transcriptomes and CUD-related changes in gene expression, we identified a subset of DETs that is regulated similarly by CUD. Our findings further support the hypothesis that cocaine-responsive genes participate in the vulnerability to substance use disorders, as we found a convergence of up-regulated DETs in individuals with CUD and genes associated with risk-taking behavior. Likewise, we found that genes linked to schizophrenia and MDD are dysregulated in CUD, indicating that these two complex disorders recruit some of the same gene regulatory and neural circuit systems. In addition, our study reveals major differences in transcriptional abnormalities within the NAc associated with CUD versus OUD based on published datasets for the latter  axis  family, EGR family, NFκB, E2F, and several nuclear hormone receptors . Drug-free age-matched control subjects (n = 20) were selected from homicides, accidental, or natural deaths that had negative urine screens for all common drugs in decedents with no history of licit or illicit drug use before death.Biospecimens were provided by the University of Miami Brain Endowment Bank, an Institutional Review Board–approved biorepository that holds frozen brain tissue annotated with demographic information and clinical variables. Postmortem male brain specimens were obtained from unaffected control subjects and from cocaine-related deaths that came to routine autopsy. Medicolegal investigations and certifications of the cocaine intoxication deaths were conducted by forensic pathologists to determine the cause and manner of death. The circumstances of death and blood and brain toxicology were reviewed to identify sudden deaths due to the toxic effects of chronic cocaine abuse in persons who met the criteria for CUD , age, race, smoking history, PMI, and brain pH to normalize factors that can affect brain RNA quality. RNA integrity numbers did not significantly differ between cases and controls (table S1). This study used male samples only because of the preponderance of males in the available brain bank. Cocaine use still predominates in males overall and the incidence of cocaine intoxication deaths follows this pattern. In addition, the rates of polydrug abuse and psychiatric comorbidity are higher among females, further excluding females based on the criteria for subject inclusion for the present study.To isolate RNA from the samples in TRIzol LS, we used the Zymo Directzol RNA Miniprep Kit . We performed deoxyribonuclease treatment on all samples to ensure the removal of genomic DNA before proceeding with library preparation. RNA was analyzed for size distribution and quantity using Bioanalyzer 2000 . We used the Takara SMARTer Stranded Total RNA-seq Library Preparation Kit to create indexed libraries from ribo-depleted RNA from each sample for sequencing . All libraries were generated simultaneously using a multiplexed library preparation strategy to ensure no variation in processing due to batch effects. Resulting libraries were analyzed for size distribution and quantity using the Agilent Bioanalyzer and TapeStation (Life Technologies) before sequencing. Molar equivalent libraries were pooled for multiplexed sequencing on the Illumina NovoSeq. We performed 2× 150–base paired-end RNA-seq and obtained a total average of 60,000,000 reads per sample. The number of independent tissue samples included in the final analysis was between 20 and 25 individuals per group. The raw FASTQ sequencing data will be made publicly available in the Gene Expression Omnibus (GEO) with accession no. GSE214267.P ≤ 0.05 and a log2FC ± 0.26  were considered differentially expressed. Chi-square test was used to examine differences in the proportion of DET biotypes between NAc and CN.After filtering, pair-wise differential expression comparisons using DEseq2 were performed to identify DETs in comparisons of control individuals versus individuals with CUD in the NAc and the CN (P ≤ 0.05), with an FDR ≤ 0.05 cutoff for significantly enriched GO terms  of molecular functions was performed using ShinyGO 0.76 with gene identities of DETs  multiplied by the sign of the FC and generates matrices of overlapped genes between RNA-seq lists of interest independent of whether individual RNAs are differentially expressed or not. RRHO was used to evaluate the overall patterns of gene expression between the NAc and CN. For comparisons of human CUD NAc and mouse self-administration NAc transcriptomes, DESeq2 was used to obtain differentially expressed genes in mouse self-administration, comparing the groups SC versus SS, CS versus SS, and CC versus SS using the datasets GSE110344 on GEO  and empirical P values were calculated, and a previously characterized threshold (Z > 10) and Bonferroni P value threshold (< 0.05) were used to assess for preservation. A similar protocol was used to separately analyze data from a previously published dataset of male mouse NAc self-administration. MDC was used to quantify differences in coexpression between CUD and controls. MDC is the ratio of the average of network connectivity between cases and controls for genes in a particular module. MDC of 2 indicates that the average correlation of genes in a module are twice as strong in cases compared to controls. MDC >1 suggests gained connectivity while MDC < 1 indicates loss of connectivity. Two separate FDR estimate were calculated by randomly shuffling samples and genes, and the final FDR was determined by selecting the larger estimate. A module was considered differentially connected is FDR < 0.01 and MDC > 2.5 or < 0.75. We used Cytoscape software platform to visually reconstruct Arachne plots . Gene expression data for all brain regions were first compiled and first covariate corrected for age, race, and PMI. The genes in the bottom quartile of variance and expression were removed. Samples with a cross-sample correlation greater than two SDs from the mean were removed. Linear relationships between gene expression were estimated by pairwise Pearson correlation. An adjacency matrix was calculated from this correlation matrix by raising it to a positive power, β, which was determined by a “scale-free” power law connectivity distribution (


Mycobacterium tuberculosis (Mtb) has been found to persist within cavities in patients who have completed their anti-tuberculosis therapy. The clinical implications of Mtb persistence after therapy include recurrence of disease and destructive changes within the lungs. Data on residual changes in patients who completed anti-tuberculosis therapy are scarce. This case highlights the radiological and pathological changes that persist after anti-tuberculosis therapy completion and the importance of achieving sterilization of cavities in order to prevent these changes.This is a case report of a 33 year old female with drug-sensitive pulmonary tuberculosis who despite successfully completing standard 6-month treatment had persistent changes in her lungs on radiological imaging. The patient underwent multiple adjunctive surgeries to resect cavitary lesions, which were culture positive for Mtb. After surgical treatment, the patient’s chest radiographies improved, symptoms subsided, and she was given a definition of cure.Medical therapy alone, in the presence of severe cavitary lung lesions may not be able to achieve sterilizing cure in all cases. Cavities can not only cause reactivation but also drive inflammatory changes and subsequent lung damage leading to airflow obstruction, bronchiectasis, and fibrosis. Surgical removal of these foci of bacilli can be an effective adjunctive treatment necessary for a sterilizing cure and improved long term lung health. Mycobacterium tuberculosis treatment has been evolving over the years, especially with the introduction of newer drugs and shorter regimens , 2. AparHere, we report a patient who underwent a complete regimen of medical therapy for pulmonary tuberculosis and later had to have surgical resection of her cavities, which grew tuberculous bacilli even after achieving sputum negativity.2. A complete blood count revealed a moderate leukocytosis of 10.2 × 109/L and an erythrocyte sedimentation rate (ESR) of 42 mm/h. Biochemical blood parameters were normal. Sputum testing found a negative acid-fast bacilli (AFB) microscopy, positive Xpert MTB/RIF test (no RIF resistance), and positive culture for Mycobacterium tuberculosis (Mtb). Additionally, drug susceptibility testing (DST) revealed sensitivity to rifampin, isoniazid, and ethambutol. Chest radiography revealed multiple small foci in the upper lobes of both lungs and a cavity in the right lung  of 16.3 kg/mA follow up chest computed tomography (CT) scan demonstrated a cavity in the right upper lobe measuring 12 × 10 mm in size with a thick and heterogeneous wall and nodules and bronchiectasis in the left lung Fig. A–D. BaseMicrobiological analysis on the resected tissue revealed acid-fast bacilli on microscopy, and positive Xpert MTB/RIF and culture results. Mtb grew from the caseous center, inner and outer walls of the cavity and a resected foci located ~ 3 cm from the cavity. DST revealed sensitivity to isoniazid, rifampin, and ethambutol.Pathological examination of the resected lesion showed findings consistent with fibrocavernous tuberculosis. No postoperative complications were experienced, and the patient reinitiated first-line therapy via DOT on the 2nd postoperative day and was discharged on postoperative day 11.A follow up CT scan performed after 3 months showed postoperative changes in the right upper lobe, and an unchanged left lung Fig. A–C. BaseMicrobiological examinations performed on resected tissue revealed positive AFB smear microscopy and Xpert MTB/RIF results and a negative AFB culture. The pathological examination of the surgical samples indicated a variety of destructive changes in addition to ongoing inflammation. The gross specimen of S1 and S2 segments of the left lung showed fibrocavernous tuberculosis shown in Fig. There were no postoperative complications, and tuberculosis (TB) treatment was reinitiated. The patient successfully completed treatment with normalization of clinical and laboratory parameters and a clinical outcome of cure in September 2021, ~ 14 months after beginning treatment. The patient had reported near complete resolution of her symptoms, having a much better ability to perform her daily activities. The patient appreciated the effects surgery had on her recovery and was happy to have gone through that treatment route. A post treatment CT scan demonstrated postoperative changes in the upper segments of both lungs Fig. D. ResultWe present this case to highlight the heterogeneous nature of pulmonary tuberculosis and need for an individualized treatment approach, especially for patients with cavitary disease. Over the last decade, novel diagnostics, drugs, and treatment regimens have revolutionized TB management including a recent landmark clinical trial demonstrating an effective 4-month regimen for drug-susceptible TB . The mov2 [Mtb has a unique ability and propensity to induce cavities in humans with various studies showing cavitary lesions in ~ 30 to 85% of patients with pulmonary tuberculosis . Lung ca2 , 16. The2 –19. The 2 .Mtb cavities are characterized by a fibrotic surface with variable vascularization, a lymphocytic cuff at the periphery followed by a cellular layer consisting of primarily macrophages and a necrotic center with foamy apoptotic macrophages and high concentrations of bacteria. Historically, each portion of the TB cavity has been conceptualized as concentric layers of a spherical structure due to its appearance on histologic cross-sections. However, recent studies using more detailed imaging techniques have shown most TB cavities exhibit complex structures with diverse, branching morphologies . A dysreThe combination of impaired cell-mediated immune responses with accumulation of inflammatory mediators at the rim of the caseum leads to ongoing tissue destruction with the potential for long-term pulmonary sequelae. Many with cavitary tuberculosis suffer chronic obstructive pulmonary disease after successful treatment and the risk may be greater in those with multidrug-resistant disease , 4. This9 per milliliter), many of which enter a dormant state with reduced metabolic activity. Bacilli in this dormant state may be less responsive to the host immune response and exhibit phenotypic resistance to some anti-tuberculosis drugs thereby preventing sterilization and increasing chances of relapse [Tuberculosis cavities not only hinder an effective immune response, but also prevent anti-tuberculosis drugs from achieving sterilizing concentrations throughout the lesion and especially in necrotic regions. The necrotic center of cavitary lesions is associated with extremely high rates of bacilli , (2) a high likelihood of treatment failure or disease relapse, (3) complications from the disease, (4) localized cavitary lesion, and (5) sufficient pulmonary function to tolerate surgery. For our patient, the severity of disease, lack of improvement of radiological imaging despite appropriate treatment, and high risk of relapse were the main indicators for surgery. Contraindications for surgery included a forced expiratory volume (FEV1) < 1000 mL, severe malnutrition, or patients at high risk for perioperative cardiovascular complications. With strict adherence to indications and contraindications for surgery, an acceptable level of postoperative complications are noted (5–17%) [Available literature points to a benefit of adjunctive surgical resection particularly among patients with drug resistant tuberculosis. A meta-analysis of 24 comparative studies found surgical intervention was associated with favorable treatment outcomes among patients with drug-resistant TB  . Additio (5–17%) . Our res (5–17%) .As our case highlights, patients with persistent cavitary disease at the end of treatment require close clinical follow up and a tailored, individualized plan to determine the best approach for disease elimination and cure. In certain cases, including those with persistent cavitary disease and end of treatment, and where available, surgical resection is an effective adjunctive treatment option that can reduce disease burden and aid anti-tuberculosis agents in providing a sterilizing cure. As we enter an era of welcomed new shorter treatment options for tuberculosis it is imperative for clinicians to be able to identify and recognize complicated TB cases that require prolonged treatment and potentially adjunctive surgery.


Dear Editor, study, publishedrecently in the Brazilian Journal of Cardiovascular Surgery, epitomizes an importantconcept of objectively quantifying the degree of haemodynamic support by computing thevasoactive-inotropic score (VIS). The authors attribute prognostic implications to apostoperative VIS >5.5 as an independent predictor of morbidity and mortality aftercoronary artery bypass grafting in their prospective evaluation.Baysal et al.. Appropriate to thecontext, Koponen et al. studydeserves a mention here which aimed to retrospectively evaluate the association betweenthe highest VIS in the first 24 hours post-ICU admission and a composite poor outcome in3,213 adult cardiac surgical patients. While their elucidation of a linear increase inthe odds of adverse primary postoperative outcome with escalating ICU-VIS scores isnoteworthy, it also does adequately emphasize the relevance of a continuous assessmentof the VIS scores extending well into the period beyond ICU admission.However, the authors’ findings need to be carefully interpreted in the absence of VISestimation in the intensive care unit (ICU) in the index study, particularly when thepredictive links of postoperative VIS are being sought with mortality and dynamic ICUmorbid outcomes, such as renal failure, central nervous damage, etc. in a small cohortof 290 patients aims at anadditional account for the prolonged haemodynamic support requirement. They describe a cumulative VIScalculation as follows: VIS0-24h(maximum) + VIS24-48h(maximum) +2×VIS48-72h(maximum), which is subsequently divided by 10 tocompute an integer VIS index. Alongside a heightened discriminative performance whencompared to the VIS (maximum) alone, a VIS index ≥3 has been outlined to beassociated with an accentuated risk of poor composite outcome after cardiacsurgery[3,.At the same time, with the understanding of the fact that VIS only allows for thehaemodynamic support quantification at a single time-point, the conceptualization of aVIS index by Crow et al.Ahead of the augmented standardization achieved by employing objective haemodynamicsupport scores like VIS, their outcome predictive potential evaluation needs to be moredeliberate with a simultaneous consideration to the two equally critical factors ofmagnitude and duration of haemodynamic support rather than envisaging a snapshot scoreconcept.


Two simpleworkup procedures that provide the pure amide product without theneed for further purification have been developed. The first employsan aqueous base-mediated annihilation of MTM. The second involvessimple product crystallization from the reaction mixture providinga low process mass intensitydirect amidation protocol.Methyltrimethoxysilane [MTM, CH However, because hydrolysisof TMOS to silica in the lung induces silicosis, TMOS is consideredfatal if inhaled (GHS H330), thereby reducing its attractiveness.Accordingly, we envisioned employing an alternative silicon-basedreagent that retains the inherent reactivity of TMOS but cannot undergohydrolysis to silica and is still amenable to removal in a workupprocedure. Herein, we present methyltrimethoxysilane  (2) as a safer  alternativeto TMOS for the sustainable direct amidation of carboxylic acids withamines  with a representativeprimary amine, a secondary cyclic amine, a secondary acyclic amine,and an aniline to enable a direct comparison with the use of TMOS(1).11 While we were hopefulthat this single “methoxy-to-methyl” switch would notbe too deleterious to reactivity, we anticipated that the workup procedurewould require significant modification to deal with the complex mixtureof linear and cyclic polysiloxanes known to result in hydrolysis ofMTM (2).12 In the event, theuse of 250 mol % MTM  in refluxing tolueneprovided pure amide products 3–10 directly aftera suitably modified workup . Specifically,evaporation of the reaction mixture postreaction removes the solventas well as siloxane (MeO)2MeSi-O-SiMe(OMe)2 andmethanol as the expected stoichiometric byproducts of the amidationprocess.13 Nonvolatile oligomeric polysiloxaneswere found to be completely removed after subsequent stirring of theresidue in a homogeneous THF/aqueous NaOH solution for 1 h, whereany unwanted methyl ester side product also undergoes hydrolysis.Any unreacted carboxylic acid is also removed in this step, and anyunreacted amine is removed in a subsequent aqueous acid wash. Thisworkup procedure thereby provides the amide products in pure formwithout the need for any further purification regardless of the extentof amidation reaction conversion.Phenylacetic acid and benzoic acid were chosen as representativeacids to be amidated using MTM (3–10 shows that MTM is as effectiveas TMOS (1) as a reagentfor amidation of the representative aliphatic carboxylic acid withall of the main amine classes  amidate malonic acid,or an α-hydroxy acid,16 to form aWeinreb amide17 or to use a low-boilingpoint amine18 under these conditions gaveamides 27–30  without the need for any further workup, thereby resultingin low PMI values as exemplified for amides 1 and 32 4.19 To t8 We therefore propose thatthese amidations take place by reversible reaction of the carboxylicacid with MTM to produce a silyl ester of type A withloss of methanol, followed by subsequent irreversible attack by amineto form the amide product  in crude reaction mixtures implicates some competitivedirect attack of silyl ester A by methanol. In supportof this mechanistic proposal, reaction of phenylacetic acid with MTM(2) showed the formation of an intermediate with a 1H NMR shift at 0.42 ppm, which is consistent with assignmentto the Si-CH3 of a silyl ester. The silyl ester was foundto be completely consumed upon addition of amine with concomitantamide formation.20Mechanistically, it has been proposed that amidationspromotedby stoichiometric silicon reagents form silyl esters as activatedintermediates. product 5. The li2)as an effective, inexpensive reagent for the direct amidation of carboxylicacids with amines, providing a safe alternative to the previouslypublished protocol using TMOS (1). The amide productscan be isolated in pure form either via a workup procedure that removesresidual MTM and any linear and cyclic polysiloxane reaction byproductsor (in the case of secondary amides) by simple crystallization fromthe reaction mixture. We expect that the latter finding will be generallyapplicable to provide secondary amides by this method with low processmass intensities.21In conclusion, we have reported the use of MTM (


Early palliative care along with standard cancer treatments is recommended in current clinical guidelines to improve the quality of life and survival of cancer patients. This study protocol aims to evaluate the effect of “Enhanced Supportive Care”, an early primary palliative care provided by nurses.N=360) and their caregivers in South Korea. Participants will be randomly assigned to the intervention or control group in a 1:1 ratio. Participants in the intervention group will receive the “Enhanced Supportive Care”, which provides five sessions of symptom management and coping enhancement counseling by nurses. The control group will receive symptom monitoring five times. The primary endpoints are symptoms, coping, and quality of life (QoL) at 3 months. Secondary endpoints are symptoms, coping, and QoL at 6 months, depression and self-efficacy for coping with cancer at 3 and 6 months, symptom and depression change from baseline to 3 months, survival at 6 and 12 months among patients, and depression among caregivers at 3 and 6 months.A randomized controlled trial (RCT) will be conducted including advanced cancer patients scheduled for first-line palliative chemotherapy (This RCT will evaluate the effects of “Enhanced Supportive Care” on symptoms, depression, coping, self-efficacy for coping with cancer, QoL and survival of patients, as well as depression of caregivers. It will provide evidence of a strategy to implement early primary palliative care provided by nurses, which may consequently improve cancer care for newly diagnosed patients with advanced stage cancer.https://www.clinicaltrials.gov/ct2/show/study/NCT04407013. The protocol version is ESC 1.0.ClinicalTrials.gov, NCT04407013. Registered on May 29, 2020,  The ultimate goal of care for advanced cancer patients is to improve quality of life (QoL). Supportive care refers to care aimed at improving the QoL of patients experiencing life-threatening diseases. Patients with advanced cancer suffer from various symptoms such as pain, fatigue, and depression, which are known to negatively affect the patient's quality of life –5.The WHO defines palliative care as early recognition of pain, physical, psychosocial, and spiritual problems faced by patients and their families related to life-threatening diseases and improving the quality of life through faithful assessment and intervention .Palliative care is not limited to the end-of-life phase. Early palliative care provided within 8 to 12 weeks of advanced cancer diagnosis demonstrated its effectiveness in managing depression and improving quality of life and survival –13. StudThe provision of early palliative care could be achieved by integrating primary palliative care into standard cancer care. Primary palliative care denotes basic features of palliative care such as basic symptom management to be delivered by any clinicians caring for advanced cancer patients , 25. CliDifficulties in accepting the term ‘palliative care’ at times of advanced cancer diagnosis have been an issue. The term ‘Enhanced Supportive Care’ has been suggested by NHS England to differentiate it from supportive care, which means the care of patients receiving active treatment  and pallSymptom management is a major element of palliative care , 30. SymNurses have played an important role in the interprofessional collaborations that provide palliative care , 22, 30.The aim of this randomized controlled trial was to compare “Enhanced Supportive Care” to symptom monitoring in terms of effects on advanced cancer patients’ symptoms, depression, coping, self-efficacy for coping with cancer, quality of life, survival, and depression in caregivers.A randomized controlled trial will be conducted by randomly allocating participants in a 1:1 ratio to the intervention or control arm. Participants in the intervention arm will receive the “Enhanced Supportive Care” consisting of symptom management and five sessions of coping enhancement counseling, while control group participants will receive symptom monitoring only. This protocol followed the SPIRIT  guidelines for reporting protocols  Patients and their caregivers will be recruited from a single cancer center that belongs to a tertiary hospital located in Seoul, Korea.Patient:Adults (age ≥ 19)Diagnosis of advanced cancerScheduled to receive first-line palliative chemotherapyEuropean Cooperative Oncology Group Performance Status ≤ 2Caregiver:Adults (age ≥ 19)Family caregivers of cancer patients who agree to participate in the studyThose diagnosed with cognitive or psychiatric problems and receiving treatmentThe random allocation sequence was developed by the principal investigator (PI) before initiating patient enrollment. Research nurses will enroll and allocate patients according to the predefined sequence. Stratified permuted block randomization will be used to assign participants in a 1:1 ratio to the intervention or control group. Strata include the type of cancer , age and sex of cancer patients. The minimization method will be applied after enrolling 60% of the participants in each cancer group to balance group allocation. Blinding is not considered applicable based on the type of intervention provided.The intervention will be “Enhanced Supportive Care”, consisting of 1) symptom management (symptom monitoring and management) and 2) coping enhancement counseling. The “Enhanced Supportive Care” sessions will be provided by nurses five times over three months. Administration of the sessions will take approximately 50 min per visit: 1) symptom management (20 min) + 2) coping enhancement counseling (30 min).Fidelity of the intervention: Web-based symptom assessment and education application, symptom management education booklet for home use, and coping enhancement counseling protocols will strengthen the fidelity of the intervention.Interventionist training and tracking: Before starting the intervention, a symptom expert (PI) will train research nurses in how to monitor symptoms and provide evidence-based symptom management education. A certified clinical psychologist will teach the principals of counseling and the third-wave psychotherapy Acceptance and Commitment Therapy (ACT) and train research nurses in coping enhancement counseling. Periodic retraining sessions will be held with a certified clinical psychologist.https://www.chartjs.org), a popular open-source JavaScript library for visualizing data. The Cancer Symptom Management system was designed so that users (researchers) had to log into access data. Patients’ symptoms will be monitored using the “Cancer Symptom Management System” on an iPad. The results from symptom monitoring will be presented as a colored bar graph to the patients’ oncologists. Anxiety and depression scores will be generated and presented along with the symptom bar graph  symptom management web-based symptom management system using iPad at each clinic visit (20 min) and 2) coping enhancement counseling (30 min). The “Enhanced Supportive Care” will be provided by trained nurses for 5 times over 3 months. Control group participants will monitor symptoms using web-based symptom management system using iPad at each clinic visits for 5 times and their symptom report will be delivered to oncologist via EMR, whereas neither evidence based symptom management education (using iPad and booklet) nor coping enhancement counseling by research nurses will be provided.Participants could withdraw from the study upon request, and those who transfer to different hospitals, whose condition is deteriorated for study participation will be withdrawn from the study. Upon patients’ withdrawal, caregiver will withdraw as well.Double data entry will ensure the integrity of the data. Collected information will be coded and stored for security, and the data will be monitored every 6 months.The data will be collected by research nurses at seven time points: before starting the first line palliative chemotherapy , on the first day of each chemotherapy from cycle 2 to 4 (T2 to T4), at 3 months (T5), 6 months (T6), and 12 months (T7) from baseline. Figure st to 4th CTx) will be monitored   + 4 corered Fig. .The primary endpoints are symptoms, coping, and QoL at 3 months. Secondary endpoints are symptoms, coping, and QoL at 6 months, depression and self-efficacy for coping with cancer at 3 and 6 months, symptom and depression change from baseline to 3 months, survival at 6 and 12 months among patients, and depression among caregivers at 3 and 6 months. Any withdrawal from adverse events related to the intervention will be recorded and reported.n=360) and their family caregivers (n=360) will participate in the study. Sample size was estimated based on two-sided tests with α=.05, power=.80 (=.93 x .93 x .93) considering three primary endpoints [Advanced cancer patients , and Mplus  software will be used for data analysis.Per-protocol analysis will be conducted for primary endpoints using data from completers at 3 months. Intention-to-treat analysis will be conducted using the imputed full analysis set as sensitivity analysis. Missing data from those who provided data other than baseline will be imputed using the last observation carried forward (LOCF) method for symptom and depression scores. SPSS Statistics , SAS , R  will demonstrate fewer symptoms and better coping and QoL at 3 months (T5) compared to the control group of advanced cancer patients who receive usual care with symptom monitoring only.Secondary hypothesis: Advanced cancer patients who receive “Enhanced Supportive Care” (symptom management + coping enhancement counseling) will demonstrate fewer symptoms and better coping and QoL at 6 months (T6), less depression, higher self-efficacy for coping with cancer at 3 months (T5) and 6 months (T6). Changes in symptoms and depression from baseline (T1) to 3 months (T5) will be different between the intervention and control groups. The intervention group will demonstrate longer survival at 6 months (T6) and 12 months (T7) than control group. Family caregivers of advanced cancer patients who receive “Enhanced Supportive Care” will demonstrate less depression than family caregivers of patients who were assigned to the control group at 3 months (T5) and 6 months (T6).Frequency and percent, mean and standard deviation (or median), minimum and maximum, and interquartile rangeStandardized difference for homogeneity for baseline characteristics between intervention and control groupsIndependent t-test to evaluate intervention effect for symptoms, coping and QoL at 3 monthsIndependent t-test to evaluate intervention effect for patients’ symptoms, coping and QoL at 6 months, patients’ depression and self-efficacy for coping with cancer at 3 and 6 months, and caregivers’ depression at 3 and 6 monthsLinear mixed model to evaluate intervention effect for change in symptoms and patients’ depression from baseline to 3 monthsPearson correlation and multiple regression analysis to identify factors contributing to symptoms, depression, coping, self-efficacy for coping with cancer and QoLKaplan‒Meier curve and log-rank test to evaluate intervention effect on survival at 6 and 12 monthsCox proportional hazard regression to identify factors contributing to survivalPearson correlation analysis, mediation and moderation analysis, and structural equation modeling to understand relationships among symptoms, depression, coping, self-efficacy for coping with cancer, and QoLThis study will evaluate the efficacy of early palliative care comprising symptom management and coping enhancement counseling provided by nurses along with standard cancer treatment in improving the symptom experience, coping and quality of life of advanced cancer patients.The study has several strengths. Early primary palliative care intervention will be provided by nurses to patients scheduled for first-line chemotherapy after an advanced cancer diagnosis following recommendations by the National Comprehensive Cancer Network (NCCN) and the American Society of Clinical Oncology (ASCO) , 30. Ear“Enhanced Supportive Care” addresses both physical and psychological management by monitoring symptoms and educating patients about self-management of symptoms from the beginning of anticancer treatment and providing counseling to enhance coping with cancer. Symptom management in “Enhanced Supportive Care” is need-based personalized symptom management education reflecting an individual’s experiences. Previous studies reported that symptom monitoring along with usual care promoted QoL and survival compared to usual care in patients with advanced cancer , 58. AccThis study will provide data on whether addressing symptom management and coping enhancement counseling by nurses can improve symptom experience, coping and QoL among advanced cancer patients.For the current RCT, participant recruitment began on 22 March 2021 and data collection will continue until 31 May 2024.


It has become well established that mitochondria not only regulate myoplasmic calcium in skeletal muscle, but also use that calcium to stimulate oxidative phosphorylation (OXPHOS). While experimental approaches have allowed for imaging of mitochondrial calcium and membrane potentials in isolated fibers, capturing the role of mitochondria and the impact of mitochondrial impairments on excitation–contraction coupling (ECC) remains difficult to explore in intact muscle. Computational models have been widely used to examine the structure and function of skeletal muscle contraction; however, models of ECC to date lack communication between the myoplasm and mitochondria for regulating calcium and ATP during sustained contractions. To address this, a mathematical model of mitochondrial calcium handling and OXPHOS was integrated into a physiological model of ECC incorporating action potential propagation, calcium handling between the sarcoplasmic reticulum (SR) and the myoplasm, and crossbridge cycling. The model was used to examine the protective role of mitochondria during repeated stimulation and the impact of mitochondrial dysfunction on ECC resulting from progressive OXPHOS inhibition. Pathological myoplasmic calcium accumulation occurred through distinct mechanisms in the model in the case of either electron transport chain, F1F0 ATP synthase, or adenine nucleotide transporter impairments. To investigate the effect of each impairment on force, a model of calcium-stimulated apoptosis was utilized to capture dysfunction-induced reductions in muscle mass, driving whole muscle force loss. The model presented in this study can be used to examine the role of mitochondria in the regulation of calcium, ATP, and force generation during voluntary contraction.The online version contains supplementary material available at 10.1007/s11538-022-01079-3. Experimental studies have demonstrated that mitochondria play a key role in modulating myoplasmic calcium during both twitch and tetanic contractions via calcium uptake mechanisms  was modeled with both voltage and calcium dependence ] and simulated with IPython 7.8.0 in Spyder v. 3.3.6 from Anaconda. The model was numerically integrated using the odeint function from the SciPy package. This function solves systems of ordinary differential equations using the LSODA algorithm Petzold , an adapFollowing action potential generation, mitochondrial calcium uptake occurred after a short delay once calcium was released from the SR Fig. d.Fig. 2 presented in Fig. An integrated model of ECC and calcium-activated OXPHOS was developed to simulate the regulatory role of mitochondria on calcium homeostasis and the consequences of mitochondrial dysfunction during sustained contractions. The model represents a first step in incorporating the effect of calcium-activated OXPHOS in skeletal muscle contraction models. Mitochondrial dysfunction was introduced via three distinct OXPHOS impairments of either the ETC, F1F0, or ANT to independently examine their effect on myoplasmic calcium and whole muscle force.Mitochondrial calcium levels peaked at approximately 50% of the myoplasmic calcium concentration during a single twitch Fig. g. This iuli Fig. a. This tuli Fig. . Once a Increased myoplasmic and mitochondrial calcium levels following muscle excitation stimulated NADH production in the model Fig. d via actAccumulation of myoplasmic calcium occurred in the model through different primary mechanisms for each OXPHOS impairment. ETC inhibition decreased the amount of calcium taken up into the mitochondria via the MCU per stimulus Fig. d, resultion Fig. , depolarDJ-1 genetic knockout found that muscle fibers presented with increased resting calcium and reduced calcium release from the SR into the myoplasm, which was attributed to a loss of myoplasmic ATP, as no change in muscle fiber excitability was observed Supplementary file2 (PDF 105 KB)Below is the link to the electronic supplementary material.


Lumbar Disc Degeneration (LDD) is associated with recurrent low back pain (LBP) (symptomatic). However, in some instances of LDD, people do not experience LBP (asymptomatic).As a step towards understanding why some people with LDD experience LBP and others do not, the primary aim of this study was to examine differences in anticipatory (APA) and compensatory postural adjustments (CPA), between symptomatic LDD patients (LDD pain) and asymptomatic LDD controls (LDD no pain) during postural perturbation. The secondary aim was to determine simultaneous differences in mental health, disability and quality of life status.U test was conducted to examine group differences in sagittal spine and lower limb kinematics , anxiety, depression, disability and quality of life.3 T MRI was used to acquire T2 weighted images (L1-S1) from LDD no pain (n = 34) and LDD pain groups (n = 34). In this observational study, responses to predicted and unpredicted forward perturbations were examined using three dimensional motion capture. A Mann Whitney The LDD pain group exhibited lower hip and knee displacements (p = 0.049−0.040) than the LDD no pain group during predicted and unpredicted perturbation. The LDD pain group also exhibited higher compensatory lumbar displacement than the LDD no pain group (p = 0.040−0.005) in the predicted condition but there was no difference observed in the unpredicted condition. The LDD pain group experienced higher levels of depression, anxiety and disability (p < 0.0001) and lower quality of life (p = 0.0001) than LDD controls.Symptomatic LDD patients are different from LDD controls; they exhibit different kinematic strategies, levels of disability, anxiety, depression and quality of life. Effective care may benefit from evaluating and targeting these differences. CurrentFunctional task analysis has been used to successfully discriminate between asymptomatic controls and LBP patients using three-dimensional motion capture (3DMC). However, findings seem contradictory and inconclusive due to the established heterogeneity of this population . In addiImpaired postural control is frequently associated with LBP . TherefoThe primary objective of this study was to examine differences in sagittal trunk and lower limb displacements between symptomatic LDD patients (LDD pain) and asymptomatic LDD controls (LDD no pain) in the APA and CPA phases of predicted and unpredicted forward postural perturbation using 3DMC. Since patients with LBP commonly experience psychological changes and disability, the secondary objective was to simultaneously examine these differences between the same groups using validated self-report questionnaires.22.1Ethical approval was granted from the NHS Health Research Authority . A priori analysis confirmed that a minimum total sample size of fifty-eight subjects (29 per group) would be required to deliver sufficient power (0.80) (α = 0.05) . Patients were recruited from primary and secondary care and healthy controls through local advertisement as part of a larger study and provided informed consent (n = 97). Since preliminary findings determined that significant differences in motor control lay between LDD pain and LDD no pain groups, these groups (sixty-eight participants (34 per group) became the primary focus of this study. LDD is common, with a prevalence of 40 % of subjects under 30 years of age, increasing to 90 % at 50–55 years . Therefo2.2A 3 T Verio MRI scanner  was used to acquire supine T2 weighted sagittal lumbar spine images (L1-L5/S1)  from healthy controls and patients as part of their consented involvement in the study and routine NHS care respectively.st and 5th metatarsals and calcanei), 3 triads   . In addiThe spine, pelvis and bilateral lower limbs were modelled as 10 rigid segments according to identifiable anatomical landmarks. The upper thoracic (UT) segment (T1-T6) was defined with its origin in T6, vertical axis from T6 to T1 (+y) and horizontal axis through T6 (+z to the right). The lower thoracic (LT) segment (T7-T12) was defined with its origin in T12, vertical axis from T12 to T7 (+y) and horizontal axis through T12 (+z to the right). The lumbar (L) segment (L1-L5) was defined with its origin in L5, vertical axis from L5 to L1 (+y) and horizontal axis through L5 (+z to the right).Pelvic, thigh, shank and foot local co-ordinate systems were defined ,31 and rThe anatomical frames of the pelvis, thigh and shank were then referenced to the corresponding technical frames in the static calibration trial such that anatomical markers  could be removed prior to dynamic trials, permitting freedom of movement. All trials were recorded at 100 Hz using a 10-camera 3DMC system (Vicon Nexus (T160), Oxford Metrics Ltd., Oxford, UK) .2) with the acceleration profile designed to simulate a train on the London Underground . Pa. Pa34]. 2.3The onsets of kinematic signals were synchronised with the onset of platform perturbation. An in-house constructed accelerometer (1000 Hz) was attached directly to the platform. The onset of perturbation was verified by a combination of visual verification and a computer algorithm reflecting the Shewart protocol . In the The baseline joint displacement was calculated within the -500 to −450 ms window prior to perturbation (0 ms). This baseline was subtracted from all outputs in order to ensure that the results were due to true differences and not differences in baseline . Since eDifferences in kinematic strategy were defined as differences in integrated sagittal spine  and/or lower limb  displacement waveforms between groups during specific APA and CPA phases. Since there is a known 50 ms electromechanical delay between the onset latency of skeletal muscle and the tension development within a muscle , each phThere was no learning effect observed with respect to the three repeated perturbation trials. Therefore, the median of three predicted and unpredicted forward perturbation trials was calculated for each participant. Three trials were excluded due to a stepping response.2.4U test, the data from both groups were automatically ordered in ascending fashion and ranked between 1 and 68. The distributions of the ranks were assessed by visual inspection of a population pyramid (histogram) produced in SPSS . Since the distributions of the two groups of the independent variable were not the same (i.e. different shapes), the Mann-Whitney U test was used to determine if there were statistically significant differences in the mean ranks of the dependent variable  between LDD pain and LDD no pain groups [Statistical analysis was performed using SPSS statistical package  and Microsoft Excel . Normality of the data was determined using QQ plots, histogram and Shapiro Wilks test. In order to perform a Mann-Whitney n groups . Higher 3Two groups were identified LDD pain (n = 34) and LDD no pain (n = 34). Age was not statistically significant between groups (p = 0.35). BMI was significantly higher in the LDD pain group than the LDD no pain group . However3.1In the predicted condition, integrated hip and knee displacements were smaller in the LDD pain group  than the LDD no pain group  . In the unpredicted condition, smaller integrated knee displacements were similarly observed in the LDD pain group (mean rank: left knee CPA2 = 27.80) when compared with the LDD no pain group (mean rank: left knee CPA2 = 37.50)  . However, in the CPA phases, lumbar displacement was notably higher in the LDD pain group  than the LDD no pain group  . In the unpredicted condition, there was no significant differences in spinal segment displacement during the APA and CPA phases (p = 0.28−0.89).These findings corresponded to a significant difference in compensatory lumbar strategy between groups in response to predicted forward perturbation with no significant difference in the unpredicted condition . There were no significant correlations in the unpredicted condition (p > 0.05).In the predicted condition, there was a significant negative correlation observed between bilateral hip APAs and self-reported pain (NRS)  than the LDD no pain group (mean ranks 17.69–24.80) . The LDD pain group had significantly lower mean ranks in terms of quality of life (mean ranks = 16.73–23.60), when compared to the LDD no pain group (mean ranks = 39.64–45.88) . This indicated that the LDD pain group experienced higher levels of depression, anxiety and disability and lower quality of life than the LDD no pain group.4The evaluation of kinematic strategy is frequently described in the literature when investigating differences between LBP and healthy controls during functional tasks . HoweverHealthcare professionals routinely use gait and STS tasks to assess LDD patients  as theseA recent systematic review, comparing anticipatory and compensatory responses to postural perturbation, found a lack of conclusive evidence to support kinematic differences between people with or without chronic LBP . DefinitIt is proposed that stereotyped lower limb displacements enhance postural recovery during mechanical perturbations on a flat surface . HoweverAlthough motor control impairments of the lower limb are typically described, significant differences in spinal strategy are documented. Healthy adults appear more adaptable than LBP patients, using an unrestricted repertoire of multiple joint segment motion in response to perturbation . HoweverAlthough, we cannot presume a causal effect, it seems that such differences in the postural chain are more likely to be uncovered through the comprehensive examination of both the spine and lower limbs. Since the postural strategy adopted by the LDD pain group involved joints proximal (lumbar) and distal (hip and knee) to the site of reported LBP, this may reflect a centrally mediated change or a change within the nervous system associated with task prediction . This reIn response to unpredictable perturbations there was no difference in lumbar and hip displacements between groups. These findings are noteworthy for two reasons. Firstly, although ‘trunk stiffening’ has been reported to result from similarly unpredictable perturbation scenarios in LBP patients , a recenA strength of this study was that the a priori sample size estimate was exceeded (34 per group). In addition, potential confounders such as age, sex, BMI and task related pain changes were not found to influence results. However, the ‘Imperial Spine’ marker set ,27 was u4.1In this study it was observed that significant differences in kinematic strategy are not exclusive to the trunk but also occur in the lower limbs. This suggests that a comprehensive examination of the trunks and bilateral lower limbs of LDD patients is required in clinical research and practice, in order to prevent the potential underestimation of motor control deficits.The kinematic differences observed between the LDD pain and LDD no pain groups in this study appear similar to those previously reported between people with LBP (for whom the cause of LBP has not been determined) and healthy controls ,49. UndeCorrelation analysis in symptomatic LDD patients revealed a negative correlation between bilateral hip APAs and self-reported pain (NRS) in the predicted condition. It is of note that, in the same predicted condition, the same parameters (left and right hip APA2) were also observed to be significantly different between the LDD pain and LDD no pain groups. A recent systematic review highlighted that the effect of LBP on APAs and CPAs is unclear, with authors proposing a link between impaired limb motor control and the risk of injury proximal or distal to the affected trunk or lower limb segments . Therefo5Symptomatic LDD patients exhibit different kinematic strategies to their asymptomatic LDD counterparts. Strategic differences are dependent upon task predictability and are not restricted to the lumbar region, confirming the need for future 3DMC studies to use marker sets, such as the ‘Imperial Spine’, which include bilateral lower limb segments. Differences in functional ability, mental health and quality of life also reflect underlying complexity. Therefore, effective rehabilitation for symptomatic LDD patients will require a multi-faceted approach. Evaluation of spine and lower limb kinematics under destabilising conditions, in addition to self-reported clinical outcomes, may prove a better indicator of LBP and/or risk of recurrence than LDD itself.The authors report no declarations of interest.