We designed a study to investigatethe coronary histological and dermal collagen ultrastructural findings in SCAD.Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndromesand in rare cases sudden cardiac death (SCD). Connective tissue abnormalities, coronaryinflammation, increased coronary n = 31 SCAD survivors and n = 16 healthy volunteerswere compared. Autopsy cases were more likely male  with greater proximal left coronary involvement  compared to SCAD survivors. N = 24(66%) of cases showed no myocardial infarction on macro- or microscopic examinationconsistent with arrhythmogenic death. There was significantly(P < 0.001) higher inflammation in cases with delayed-onset deathvs. sudden death and significantly more inflammation surrounding the dissected vs.non-dissected vessel segments. N = 17 (47%) cases showed limitedintimal fibro-elastic thickening but no features of fibromuscular dysplasia and noendothelial or internal elastic lamina abnormalities. There were no differences in VVdensity between SCAD and control cases. TEM revealed no general ultrastructuraldifferences in ECM components or markers of fibroblast metabolic activity.Thirty-six autopsy SCAD cases were compared with 359 SCAD survivors. Coronary andmyocardial histology and immunohistochemistry were undertaken. Transmission electronmicroscopy (TEM) of dermal extracellular matrix (ECM) components ofAssessment of SCD requires careful exclusion of SCAD, particularly in cases withoutmyocardial necrosis. Peri-coronary inflammation in SCAD is distinct from vasculitidesand likely a reaction to, rather than a cause for SCAD. Coronary fibromuscular dysplasiaor increased VV density does not appear pathophysiologically important. Dermalconnective tissue changes are not common in SCAD survivors.  Giudicessi andSharonne N. Hayes, Spontaneous coronary artery dissection (SCAD), especially of distal coronary territoriesshould be carefully assessed at post-mortem in SCD cases, even where there are no signs ofmyocardial infarction. The immediate cause of SCAD is likely to be the development of aspontaneous intramural haematoma rather than an intimal disruption or ‘tear’. This does notseem to be directly related to increased VV density, coronary fibromuscular dysplasia, orlocal inflammation (except as a response to injury). Although SCAD is rarely associated withhereditary connective tissue disorders, there does not seem to be a more generalizableglobal connective tissue ultrastructural abnormality in most cases.2 In rarecases, the first clinical manifestation is with sudden cardiac death (SCD). These cases willpresent at autopsy although diagnosis can be challenging and SCAD may be under-representedin the autopsy series of SCD.3Spontaneous coronary artery dissection (SCAD) is an uncommon cause of acute coronarysyndromes. Clinical presentation is usually with acute myocardial infarction, which may beassociated with ventricular arrhythmia in 3–10% of cases.3 This leads to alongitudinal dissection plane causing external compression of the true lumen. Twomechanistic hypotheses have been proposed. The inside-out hypothesis suggests anendothelial-intimal disruption (‘dissection flap’) as the primary event allowing blood toenter the sub-intimal space, whereas the outside-in hypothesis suggests the primary event isa de novo intramural bleed.4 One clinical imaging study reported an increase in vasavasorum (VV) density as a potential source for an intramural bleed,5 although a subsequent larger study didnot replicate this finding.6 A rolefor inflammation8 and abnormalities of connective tissue9 in the pathophysiology of SCAD has alsobeen proposed. Indeed, inflammatory cell infiltration of the peri-adventitial tissuesurrounding the affected coronary has often been described as a component of thehistopathological picture of SCAD in autopsy reports, although the relationship to the SCADevent is unclear.10 Inflammatorydisorders and hereditary connective tissue disorders are reported in SCAD survivors,although precise mechanisms are not known.11Descriptions of the range of histopathological findings in SCAD have been limited toisolated case reports and small series. SCAD is reportedly characterized by the presence ofan intramural thrombus within a false lumen in the tunica media of the affected coronaryartery.In this study, we aimed to investigate the spectrum of histological findings of SCAD fromthe largest reported autopsy series assembled to date and to study the dermal collagenultrastructure of SCAD survivors to explore the implications of these findings both forclinical pathology and our understanding of the pathophysiology of this condition.The study was conducted according to the principles of the Declaration of Helsinki.Autopsy-diagnosed SCAD cases who were referred with SCD were identified retrospectivelyfrom international cardiovascular pathology centres: the UK CRY (Cardiac Risk in theYoung) database ; theVictorian Institute of Forensic Medicine ; and the Universityof Utrecht Medical Center . Ethical approval for this study wasgranted in the UK (REC reference is 10/H0724/38), the VIFM [in accordance with section2.3.5 of the NHMRC National Statement on Ethical Conduct in Research and the conditionsset out in the Coroners Act 2008 (Victoria)—reference EC01/2017], and the Netherlands [thestudy and coding of human material met the criteria of the Netherlands Code of Conduct forthe responsible use of human tissue for medical research as approved by the local BiobankReview Committee of the University Medical Center Utrecht (protocol number 15-252)]. Age-and sex-matched controls were identified from the CRY archives as individuals who sufferedpresumed sudden arrhythmic death syndrome (SADS) with a morphologically normal heart atautopsy, without evidence of SCAD or other obvious pathology. All cases underwent eithercoronial autopsy in accordance with a legal practice appropriate to the relevant nationaljurisdiction or routine autopsy for which consent was given by their next of kin.Permission for further evaluation of material from the autopsies without informed consentwas provided by the relevant ethics committee as detailed above and in accordance withrelevant national legislation for the handling of human tissue.Consecutive SCAD cases and healthy volunteers (HV) were recruited to the UK SpontaneousCoronary Artery Dissection (UKSCAD) Study (ISRCTN42661582). Patients with SCAD arerecruited from across the UK by self-referral, primary care physician referral, andreferral from the clinical team at the index presenting hospital. HV were defined asindividuals aged 18 and above who have never been diagnosed with any chronic condition anddo not take any regular medications . All participantsprovided fully informed signed consent. The protocol was approved by the UK HealthResearch Authority (14/EM/0056).FigureS1.A visual summary of the study design and different study populations/groups is shown inN = 27 autopsy cases had sufficient detailsregarding the circumstances of death and prior symptoms described by the individual, basedon which the time period between onset of symptoms and death could be determined as<24 h (defined as ‘rapid-onset death’) or ≥ 24 h (‘delayed-onset death’). For the SCADcases and HV population, demographic information, medical history, and a detailed historyof the SCAD event (if applicable) were obtained from medical records and directly from thesubject. Pregnancy-associated SCAD (P-SCAD) was defined as SCAD occurring during gestationor within 12 months of delivery. Hypertension and dyslipidaemia were defined by the needfor active treatment prior to SCAD.For autopsy cases, demographics, circumstances of death, clinical data, and macroscopicfindings for each case were obtained from the coroner referral letters and reports.Information was sought on previous cardiac signs/symptoms, history of pregnancy orpost-partum at time of death, family history of connective tissue disorders or SCD, drug,and other medication use. Haematoxylin and Eosin (H&E), as well as Elastic Van-Gieson (EVG)-stained sectionsof culprit and non-culprit coronaries, were examined under light microscopy. All casesexamined had at least one H&E-stained coronary artery section from the left mainstem (LMS), as well as one of the proximal and one of the distal part of the threecoronary arteries in addition to sections of the site of dissection.n = 20 SCADcases, where blocks of the SCAD-affected coronary artery were available. As a comparatorcontrol group, we randomly selected N = 10 age- and sex-matched SADScases from the CRY archive. Sections were cut from paraffin-embedded tissue and fixed onSuperFrost slides. IHC was undertaken for the following targets: CD68 ; CD3 ; CD31 , α-smooth muscle actin ; CD34 . Staining was conducted on a Ventana Benchmark Ultra autostainer asper the manufacturer’s guidelines, using the Optiview DAB kit for detection of targets.To quantify VV density in the vasculature, IHC was undertaken for CD31 . VV was quantified by counting the number of CD31+stained vascular structureswithin the media and adventitia of the coronary sections. Adjustment for vessel size wasperformed by dividing the number of VV by the maximal diameter of the coronary sectionstudied. Additional analyses were undertaken by quantifying the total CD31+ stainingwithin the tunica media of the coronary.12 Staining for CD34, expressed on the surface of endothelialprogenitor cells and mature endothelium, was used as a comparable positive control. Allimage analyses were performed using ImageJ software.13Immunohistochemistry (IHC) was performed in a subset of 15 A percentage wasthen calculated for each feature observed in each subject. All analysis was conductedblinded to SCAD and HV status.Dermal skin biopsies were collected, after local anaesthesia, through resection of asmall ellipse (5–6 mm) on the inner side of the left upper arm. Skin biopsies werecollected in 5 mL of cold filtered 2.5% glutaraldehyde solution  diluted in phosphate buffer (PB) 0.1 M . Each fixed tissue was then cut longitudinally into three smaller pieces of 1 mmwidth and further fixed in glutaraldehyde 2.5% solution from 2 h up to overnight. Tissuesections were then washed in PB 0.1 M and secondarily fixed in 1% osmium tetroxide  dissolved in potassium ferricyanide 1.5%  for90 min. Sections were then washed in distilled de-ionised water, dehydrated through aseries of 70%, 90%, and 100% analytical grade ethanol , and processedin a gradually increasing concentrations of epoxy resin dissolved in propylene oxide . Finally, the specimens were oriented inorder to show the full thickness of the dermis and embedded in pure resin. Using a LeicaUM UC7 microtome, the resin blocks were trimmed to thin sections (70 nm) and placed oncopper grids . Sections were counterstained using 2% uranyl acetate, followed by the immersion in a lead citrate solution, anin-house solution obtained by mixing lead nitrate  and sodiumcitrate . The sections were viewed with a JEOL JEM-1400 transmissionelectron microscope (TEM) with an accelerating voltage of 100 kV using an OlympusMegaview III with iTEM software digital camera. Images of collagen fibrils, elastin, andfibroblast cells were all taken in the mid-reticular dermis. Collagen fibrils minimumdiameter was measured in at least 5–6 images of transversal section orientation in atleast three different collagen bundles. The measurements were performed by using a macrodesigned with the ImageJ software that allowed to measure about 2000–3000 fibrildiameters per subject. Images of collagen fibrils in transverse sectional orientationwere also examined for the presence of irregular fibrils (fibrils with irregular edges)in 8 to 10 different collagen bundles distributed in 4 to 5 areas of the copper grid. Anaverage of 8 images of elastin and fibroblasts per subject were also taken and thewidest diameter in each image, as well as for the diameters of irregular fibrils, wasmeasured by post-analysis of the images with ImageJ. Elastin images were qualitativelyanalysed for the presence of features reported in hereditary connective tissue disorderssuch as frayed edges, moth-eaten edges, calcified microcavities, dense internal spots,thick surface coat, and indentations.Summary statistics are provided for all variables, including means ± standard errors forcontinuous variables and counts and percentages for categorical variables. Continuousvariables were tested for normality using Kolmogorov–Smirnov test, and by visualinspection of the data.t-test or Mann–Whitney U-testand Fisher exact tests, respectively.When comparing the UKSCAD cohort with the autopsy cases, as well as in the histology andIHC experiment analyses, differences in continuous and categorical variables were testedusing independent sample P-values. A P-value of <0.05 was consideredstatistically significant. All statistical analyses were performed using SPSS version 20.0or GraphPad Prism version 8.0.In the electron microscopy experiments, univariate and multivariable regression analyseswere carried out to test the impact of the most relevant clinical data  on thequantitative and qualitative variables measured in the collagen fibrils, elastin, andfibroblasts. For continuous outcomes linear regression was performed and mean differencespresented. For count outcomes Poisson regression was employed and incident rate ratiospresented. For binary outcomes, logistic regression was performed and odds ratiospresented. All estimates are presented with 95% confidence intervals (CIs) andn = 29females (81%) with an average age of 49.4 ± 2.5 (range 26–78 years). Three of the patientswere P-SCAD, accounting for 10% of females in the case series. The prevalence ofcardiovascular risk factors is shown in Table 1. None of the cases had a known inflammatorydisorder, connective tissue disorder, or relevant family history.A total of 36 autopsy cases of SCAD were studied from 1996 to 2016. None of the cases hada diagnosis of SCAD prior to death. The case series consisted of Table1). Although in both groupsmost patients were female, there was a significantly higher percentage of femaleindividuals in the UKSCAD cohort compared to the autopsy cases . The percentage of P-SCAD cases was similar between thetwo groups. Autopsy cases had higher BMI (P = 0.0290), higher prevalenceof active smoking (P = 0.0014), and dyslipidemia(P = 0.0114).The demographics and risk factors were compared with the UKSCAD cohort . Localization ofSCAD in the autopsy series was compared with angiographic data from the SCAD-survivorcohort (Table 2). Therewas a significantly higher proportion of SCAD autopsy cases involving the LMS or proximalcoronary artery segments compared to SCAD survivors . These included n = 3 casesoriginating in the LMS but also extending into the left anterior descending (LAD) artery.There were significantly more SCAD cases involving the LAD, predominantly in themid-distal vessel . In the autopsy case series, the extent of the dissectionalong the length of the culprit vessel varied from 5 to >50 mm.All autopsy cases involved a single coronary artery , there was no evidence ofnecrotic myocardium on either macro- or subsequent microscopic examination. Of the threeP-SCAD cases in the autopsy series, two originated from the proximal LAD and one from thedistal left circumflex (LCx), extending into the posterior descending artery and incontrast to non-P-SCAD autopsy cases, all P-SCAD cases were associated with necrosis ofthe underlying myocardium corresponding to the affected vessel.In two-thirds of the autopsy cases  of autopsy cases had minimalinflammatory cell infiltrate, whilst two-thirds of the cases (n = 24)displayed significant infiltration with neutrophils/macrophages, lymphocytes, and/oreosinophils in the adventitia with varying degrees of extension into the medial layer.N = 11 of those cases showed fully organized peri-adventitial fibroustissue. There were only very rare giant cells around the elastic fibres of the dissectedsegments. In all cases, the inflammatory cell infiltrate was limited to sectionscontaining the false lumen and was not present in healthy, non-dissected, proximal ordistal sections of the culprit and non-culprit coronaries.There was substantial heterogeneity in the degree of inflammatory cell infiltrate inthe sections studied. One-third . In SCAD cases with significant inflammation,there was abundant CD68+ staining throughout the adventitial infiltrate, extending intothe perivascular adipose tissue and in the media surrounding the dissection plane andhaematoma .Similarly, there was significant, albeit less pronounced staining for CD3, whichappeared to be more spatially localized over the adventitial border of the vascularwall, as well as the outer rim of the media and adventitial inflammatory infiltrate.To compare the SCAD inflammatory cell infiltrate with the established histopathology ofmedium- and large-vessel arteritides, we performed IHC for CD68 (surface marker ofmacrophages) and CD3 (surface marker of T-lymphocytes) and compared with age- andsex-matched control cases. There was, as expected, significantly higher infiltration ofCD68+ and CD3+ cells in the peri-adventitial tissues surrounding an SCAD sectioncompared to control cases  and CD3+showed a similar significant link between abundant cellular staining and longer timeinterval from symptom onset to death.We next assessed the association between time elapsed from symptom onset to death anddegree of inflammatory infiltrate. Two researchers, both blinded to clinical details,independently analysed n = 18 autopsycases that exhibited high inflammatory infiltrate, there was a significantly largersurface area of peri-adventitial reactive tissue adjacent to dissected vs. non-dissectedsegments of the coronary sections examined, after adjusting for the percentage of totalvascular circumference affected . Furthermore, in H&Esections, the number of peri-adventitial inflammatory cells surrounding the dissectedsegment was significantly higher compared to the non-dissected segment, adjusted for thepercentage of vessel circumference affected by the dissection .These results were replicated when examining the number of CD68+ macrophages and CD3+ T-lymphocytes corresponding to the dissected vs. non-dissected segment in IHC analysis.In addition to this temporal association, we sought to establish a spatial associationbetween inflammation and the dissected media. In the Figure ). Total CD31 staining  in the media and adventitia also did not differ betweenthe two groups . When comparing SCAD autopsy cases with rapid- vs.delayed-onset death, we observed a trend towards denser VV  and more abundant medial andadventitial CD31 staining , although the association did not reach statisticalsignificance. The distribution of CD34 staining was similar, confirming the structuresstained as VV .After adjusting for vessel diameter, no significant differences in the density of VVwere found between SCAD sections and controls , the dissection event occurred near theouter media, close to the adventitial border. In the remaining cases, the medialintramural haematoma was localized close to the internal elastic lamina (IEL).In most cases ; on the other hand, more proximal sectionsbelonging to the same case displayed a false lumen enveloping almost the entirety of thecoronary circumference .The proportion of the total coronary circumference affected varied widely both withinand between cases. Some sections displayed a small intramural haematoma accounting forless than 10% of the vessel medial area . Other caseshad varying degrees of mixed layers of fibrin formation, with almost distinct‘compartmentalization’ of different segments of the intramural haematoma, giving a‘trabeculated’ appearance, akin to the so-called lines of Zahn seen in fresh thrombus.The appearance and constituents of the false lumen were heterogeneous. Some casesdisplayed dense red clot with trapped white cell nuclei and minimal fibrin formation of the autopsy cases studied displayedsome intimal changes. These ranged from mild to moderate thickening (a recognizedfeature of ageing) but also included changes consistent with underlying atherosclerosis.Approximately half of the cases  had mild to moderateatherosclerotic changes in both culprit and non-culprit coronary arteries. These changeswere mostly limited to early neo-intima formation, with proliferation of vascular smoothmuscle cells and sometimes the formation of foam cells in the intima. Only one casedisplayed significant atheroma (80% stenosis) on macroscopic examination, but in anon-dissected coronary vessel.More than half . To further confirm the presence of intact endothelial cells, we performedIHC staining for CD31 (PECAM-1). Staining for this mature endothelial cell marker showedthe normal presence of endothelial cells in the intima of all histological sectionsstudied .EVG staining also did not reveal evidence of excessive collagen deposition in the intimaor the media, which is distinct from the mild fibro-elastic intimal thickening seen insome cases . In addition, IHC for α-smooth muscle actin showed a normal patternof staining across the non-dissected segments of the media in SCAD sections .No recognized histological features of fibromuscular dysplasia (FMD) were identified.FMD is characterized by thickening and proliferation of the intimal layer andobliteration of the medial layer through extensive, dense, deeply stained collagendeposition, as well as fragmentation of the internal and/or external elastic lamina.These features are prominent in the example provided in Figure ).We did not observe evidence of IEL degradation or fragmentation in any of the sectionsstudied or differences when compared to control cases ; fibroblastsand their subcellular synthetic organelles ; or features of elastin damage  between SCAD cases and HV.Dermal connective tissue from 31 patients and 16 HV was assessed by trasmission electronmicroscopy . Demographic characteristics and cardiovascular risk factors can be found inTablesS1 and S2. This demonstrated asignificant effect of age on minimum collagen fibril diameter(P = 0.0011), the number of irregular fibrils(P = 0.015), and elastin calcified microcavities(P = 0.0031). The number of elastic calcified microcavities was alsosignificantly different (p = 0.0046) between those with low and highbeighton score. In addition, significant differences between SCAD and HV were found forelastin thick surface coat (P = 0.0285) and elastin calcifiedmicrocavities (P = 0.0491).Univariate and multivariable analyses are presented in We present the largest study to date of SCAD coronary histopathology and the firstsystematic assessment of dermal collagen ultrastructure in SCAD survivors. We report,firstly, that myocardial necrosis is absent in a majority of autopsy cases suggesting arapid or arrhythmic death. Secondly, inflammatory infiltration develops over time and likelyconstitutes a healing response to injury. Thirdly, we find no evidence ofendothelial/intimal injury, no coronary histological features of FMD, and no evidence of anincreased VV density in SCAD. Finally, we show no ultrastructural differences in dermalcollagen and no evidence of changes in the cellular activity of skin fibroblasts in SCAD.Nevertheless, some features of elastin damage do appear to significantly differ between thetwo groups.3 The finding that P-SCAD was associated with moreextensive myocardial infarction requires validation but is consistent with a number ofstudies reporting P-SCAD is a more extreme phenotype.17 This,coupled with the recent demonstration that post-menopausal women with SCAD may have a morebenign phenotype than pre-menopausal women,18 suggests changes in female sex hormones may have a role in determiningthe severity of SCAD at presentation, although the mechanism remains unclear.The study findings have important implication for the autopsy assessment of SCD. Thepresence of more proximal dissections when compared to patients surviving to angiography isconsistent with higher-risk cases leading to fatality. However, the lack of myocardialinfarction in a majority of cases suggests many deaths are arrhythmic and an absence ofmyocardial necrosis cannot rule out this diagnosis. These findings also suggest somepatients with shorter, more distal SCAD presenting with arrhythmic death may be missed atautopsy unless this diagnosis is carefully excluded by systematic assessment of the entirecoronary tree, as suggested by a previous smaller case series.19 These findings taken together aresupportive of the outside-in hypothesis as the predominant mechanism for SCAD. Additionally,coronary microvessels have been proposed as the potential source of intramural bleeding inSCAD. A previous intracoronary imaging study reported an increase in VV density in SCADcoronaries5 although this wasnot confirmed in a subsequent larger series.6 This histological study confirms no evidence for increased VV densitysuggesting that absolute vessel density may be less important than the vulnerability oftraversing microvessels and the disrupting forces to which they are subjected. It has beendemonstrated that adventitial vasoactive factors play an important role in regulatingcoronary arterial tone leading to speculation of an additional potential element to theoutside-in pathophysiological hypothesis of SCAD.20A number of hypotheses as to the arterial vulnerability and pathophysiological mechanismunderlying SCAD have been proposed, to which our data provide novel insights. Although itwas not possible to serially section the entire coronary tree of affected patients, nostructural abnormalities of the coronary intima or the IEL were demonstrated, as might beexpected for a spontaneous tear to develop (as implied from the inside-out hypothesis).Intracoronary imaging has provided evidence of false lumen pressurization prior to thedevelopment of fenestrations between false and true lumens and angiographic findings haveshown these fenestrations occur after the development of intramural haematoma and not as apre-requisite for SCAD.2 The exact proportion of SCAD cases with extra-coronary arteriopathies isunclear due to variations in the definitions and imaging modalities used in differentstudies.21 One recent study even reported 100% of SCAD cases hadradiological arterial ‘abnormalities’ of some sort.22 This has led to speculation that SCAD arises primarilyas a complication of pre-existent coronary histological FMD.23 In this study, the typical histopathological features ofFMD were not seen in the coronary artery sections studied, suggesting that changes oflocalized coronary histological FMD are not a pre-requisite to SCAD in many cases. It istherefore likely that other, more subtle changes in the coronary vessel wall, such asdifferences in cell–cell adhesion or extracellular matrix function, are responsible for thevulnerability to SCAD. Histological FMD was not found on a limited review of availablenon-coronary arterial material from the autopsy cases. This may represent incompletesampling but it remains to be confirmed that the ‘string-of-beads’ appearance ofradiological FMD seen in SCAD invariably corresponds to histological changes consistent withthe pathological definitions of FMD. The only reported post-mortem case presenting the grosspathological appearances of a renal artery ‘string-of-beads’, does not describe thehistological findings of this artery.24 In this study, we are unable to definitively address the question as towhether the coronary histology of patients with SCAD and extra-coronary arteriopathies differs from SCAD cases without sucharteriopathies. A future prospective series with systematic serial sectioning of relevantarterial beds will be required to address these questions definitively. The low rates ofatherosclerotic changes seen in the autopsy cases may reflect the low-risk profile of thispredominantly female population but are also consistent with recent findings suggesting anopposing influence of common genetic variants on SCAD vs. ischaemic heart diseaserisk.26A significant proportion of patients with SCAD have been shown to have co-existent remotearteriopathies, particularly the ‘string-of-beads’ sign of radiological FMD.8 Our studypresents the most comprehensive evidence so far supportive that coronary inflammation inSCAD is a time dependent and localized healing response to the injury rather than a causalvasculitic process. This inflammatory infiltrate is distinct from that of medium- andlarge-vessel arteritides: There were scarcely any giant cells noted, a predominant featurein giant cell arteritis (GCA).27The predominant cell type was CD68+ macrophages, as opposed to GCA and Takayasu’s arteritis,where the infiltrate is primarily CD3+ T-cell abundant.27 Eosinophilic infiltration was not a consistent globalfeature across the SCAD case series as described in eosinophilic coronaryperiarteritis.28 Our findingsare consistent with the fact that, although inflammatory disorders are often reported as apredisposing condition in SCAD,11rates of inflammatory diseases are probably similar to the general population.30Previous histopathological case reports have described SCAD as a mono-arteritis because ofthe density of the reported associated inflammatory infiltrate.32Features of hypermobility have also been described in a subgroup of SCAD survivors. This hasled to speculation that even without a monogenetic cause, abnormalities of connective tissuemight be a common mechanism underlying SCAD.2 Abnormalities of dermalcollagen ultrastructure have been shown in a range of established connective tissuedisorders.15 We found nogeneralizable difference in a range of connective tissue ultrastructural features on blindedanalysis. Importantly, previously reported effects of age33 on collagen fibril size and the number of irregularfibrils were confirmed, providing effectively a positive control within the analysis. Somefeatures of elastin damage were different between HV and SCAD survivors, suggesting apossible underlying predisposition towards more unstable elastin in these patients. Thesefindings are hypothesis-generating and will require further validation but are in keepingwith recent genetic studies34 showing causal connective tissuedisorder variants in SCAD affect only <5% of index cases and suggests ultrastructuralchanges in dermal connective tissue are not a common feature in SCAD. Future assessment indemographic and genetic subgroups will be of interest.SCAD is associated with hereditary connective tissue disorders in a small proportion ofcases.SCAD leading to SCD is rare, thus making a prospective unbiased design logisticallyimpossible. As a retrospective observational study, we cannot conclude that theassociations demonstrated are causative. All SCAD autopsy cases were initially referredfor clinical autopsy and as such, it was impossible to employ a uniform methodology orsequential sectioning of the entire length of the coronary tree. Our ethical permissionsdid not permit genetic analysis of autopsy cases. As most autopsies are initiallyperformed by non-cardiovascular pathologists and the heart (only) is retained for laterexamination by a cardiovascular pathologist, limited non-coronary arteries were availablefor assessment. Interpretation of the frequency of non-coronary arteriopathies istherefore limited by incomplete sampling. The numbers of included patients will impact onpower meaning small effects in the measured indices may not be demonstrated. Dermalconnective ultrastructure was used as a surrogate for coronary connective tissue astrasmission electron microscopy could not be undertaken on the dissected coronaries, giventhat fresh tissue is required.Care is required during autopsy for SCD to exclude SCAD, particularly when affecting moredistal coronary locations and presenting without myocardial necrosis. This study found nosupporting evidence for a causal role for peri-coronary inflammation, which is more likelyan evolving response to injury than a coronary mono-arteritis triggering an SCAD event. Wealso found no evidence to support the inside-out hypothesis, no features of coronary FMD,and no evidence for an increased VV density as the primary source for intramural bleeding.Finally, we find no generalized changes in dermal collagen ultrastructure, suggesting thesechanges may not be a prime pathophysiological driver in most patients.Cardiovascular Research online.All authors contributed to the design of the work, interpretation of the data and the finalwriting of the manuscript. M.M., F.S., and A.A.B.-C. additionally contributed to theacquisition and analysis of the data and C.B. to the statistical analysis of the data. D.A.and M.N.S. are accountable for all aspects of the work and undertake to ensure thatquestions related to the accuracy or integrity of any part of the work are appropriatelyinvestigated and resolved.cvab183_Supplementary_DataClick here for additional data file.


Bacillus subtilis bactofilins BacE and BacF were shown to be important for swimming motility in strain PY79, and single gene deletions were reported to be lethal, in contrast to a double bacEF deletion.Bactofilins can assemble into polymeric structures and play important roles in cell shape maintenance, chromosome segregation and motility. B. subtilis strains, with strain 168 showing no defect in motility, and 3610 showing delayed induction of swimming. Generation of single gene deletions in PY79 was possible by transferring corresponding deletions from 168. In the natural isolate 3610, gene deletions also showed a negative effect on biofilm formation, revealing an additional function for BacE and BacF. A spontaneous arising suppressor mutation in PY79 was mapped to the flhO gene, a constituent of the flagellum, which obtained an 18 amino acid extension at its C-terminus. Our findings show that bactofilin gene deletions lead to different motility phenotypes dependent on the strain background, and affect biofilm formation in the natural isolate 3610. Our data reinforce the idea of a connection between bactofilins and motion via the flagellum, and suggest that they operate in a switch like manner.Extending this work, we show that motility defects vary between different The online version contains supplementary material available at 10.1186/s13104-022-06048-6. Thermus thermophilus bactofilin TtBacA recently suggested a head-head polymerization mechanism and TtBacA was also found to exhibit intrinsic membrane affinity [Bactofilins are a widespread class of small filament-forming proteins. Homologs have been found all around the bacterial phylogenetic tree and are frequently duplicated within genomes , 2. AddiCaulobacter crescentus that is essential for stalk formation [Myxococcus xanthus bactofilins BacNOP that were shown to interact with the chromosome segregation machinery [Helicobacterpylori, which acts in line with a dynamic peptidoglycan (PG)-modifying machinery that locally changes the cross-linking pattern in the PG-sacculus and thus enables the typical cork-screw like shape of the bacterium [Among the best studied examples are BacA from ormation , and Myxachinery . Anotheracterium .B. subtilis possesses two bactofilin homologs, BacE  and BacF (yhbF) [bacEF by a tetracycline resistance cassette resulted in loss of swimming motility in PY79. Further investigation unveiled that the cells were not able to generate neither the flagellar hook nor the filament [The gram-positive model organism F (yhbF) . Using fB. subtilis, assembly of functional flagella is a time and energy costly process that involves over 20 structural proteins and underlies a complex assembly-order. It is still unclear how bactofilins may act on the formation of the flagellum, or the regulation thereof. B. subtilis lab strains exhibit two forms of active, flagella driven movement, swimming and swarming [Flagella-mediated motility is a life-saving feature for many bacteria, enabling them to survive in environments that are dominated by nutrient gradients. Due to its drastic influence on the lifestyle of swarming , 9. Swimswarming .bacE or bacF single knock-out strains failed in our earlier study. Both genes could only be deleted in the presence of an ectopic copy of both genes, suggesting that the loss of single genes is lethal [bacE and bacF mutants in the Bacillus-genetic-stock-center  deletion collection [comIQ12L mutation [bacF-mutant colonies in strain PY79 frequently reverted to swarming cells after extended cultivation on solid media. Complementing this work, we created a suppressor mutant of PY79 bacEF::tet that is as motile as its wildtype progenitor and found a single nucleotide deletion in the putative rod-gene flhO as causative reason. This work clarifies and extends results found in El Andari et al. [All attempts of creating s lethal . To our llection   deletion resulted in cells of the lab-strain PY79 lacking filaments and hooks, and the construction of single bactofilin knock-out mutants failed. Motility in the bacEF double mutant strain could not be complemented by ectopically expressing either of these genes, only the expression of both proteins was able to restore motility. However, within the B. subtilis mutant strain collection [bacEF double mutant in 168 by using genomic-DNA from PY79 bacEF::tet, and analogously, single and double gene deletion strains in the undomesticated strain 3610 , allowing cells to use flagellar motion to move on semi-solid agar of 0.5%. This so-called swarming motion allows for spreading also on higher concentrated agar plates as shown in Fig. bacE or bacF mutant cells did not show any swarming motility , unlike wild type cells, which formed structured colonies . We used the alpha-fold algorithm [B. subtilis FlhO structure  for a bacEF deletion in PY79 does not mean that there could be other mutations, which might compensate for the lack of BacF. These could also appear in structures other than the flagellum. Delay of swimming activity seen in bacF mutant cells in strain 3610, and arising of suppressors is highly stochastic, so this effect is highly variable and difficult to (Additional file The identification of a single suppressor (Additional file1 Table S1: List of primers used in this study


Dear Editor,1 Several cutaneous manifestations can be seen in the disease course. Discoid lupus erythematosus (DLE) is the most common type of cutaneous lupus erythematosus. It may be associated with an increased risk of cutaneous squamous cell carcinoma (cSCC) that may have a poorer prognosis in cases with particular risk factors. The aim of this manuscript was to share our patient with SLE presenting cSCC as a complication of DLE and to draw attention to these risk factors in such cases.Systemic lupus erythematosus (SLE) is a multisystem disease with a large range of manifestations.A 50-year-old woman with a history of SLE and DLE for 8 years has presented a non-healing cutaneous ulcerated lesion over the discoid area on her nose . Due to 2 High-risk skin cancers are a rare, but severe, complication of DLE. Cutaneous squamous cell carcinoma has been reported with an incidence up to 3% of patients with DLE.2 Cutaneous squamous cell carcinoma may present different clinical behaviors ranging from relatively non-aggressive forms to aggressive ones with significant metastatic potential, but scars and immunosuppression have been reported as predisposing factors associated with high-risk subtypes.3 Previous studies have reported poorer data in cSCC associated with DLE with higher rates of recurrence and metastasis than sporadic cSCC cases.4 Also a 4-fold higher risk of cSCC development has been reported in patients with DLE which is associated with a 20-fold increase in mortality compared to patients with sporadic cSCC (2). So the clinical course of cSCC as a complication of DLE may be aggressive, with early metastases and risk of mortality.6Discoid lupus erythematosus is characterized by scarring lesions with pigmentary changes mostly on sun-exposed skin areas.7 Long-standing discoid lesions seem to be one of the major factors in cSCC arising in DLE since previously described cases generally presented long-term DLE history as in our case.6 Also UV radiation is a well-known risk factor for cSCC and DLE lesions located preferentially on sun-exposed body areas.2 Disease-related immune system impairment and cutaneous inflammation are also suggested as possible pathogenetic mechanisms.7 Immunosuppressives have been considered as factors promoting cSCC development; however, similar cSCC prevalence was reported between DLE patients with or without immunosuppressive drug history (2). Although the underlying pathophysiology has not been clarified yet, DLE-associated cSCC seems to be reasonable in patients with ACTDs because of several cumulative risk factors in this disease spectrum. Also, it has been stated that the clinical presentation may be challenging and the cSCC risk airing in DLE can be minimized by early anti-inﬂammatory treatment.9Although the pathophysiology underlying the development of DLE-associated cSCC has not been defined, several factors may cause predisposition to skin cancer in DLE. Cutaneous malignancies have been significantly associated with autoimmune connective tissue diseases (ACTDs) including SLE, systemic sclerosis, dermatomyositis, and Sjögren syndrome due to several potential pathogenetic mechanisms such as chronic scar, ultraviolet (UV) radiation, inflammation, immunosuppressive treatments, viral infections, and smoking.In conclusion, cSCC, one of the most common form of non-melanotic skin cancer, may develop as a complication of DLE, especially in patients who have additional risk factors due to the nature of the co-incident diseases such as ACTDs. The poorer prognosis of cSCC in DLE patients highlights the need for better screening strategies for early detection and prompt therapeutic interventions.


Post-stroke cognitive impairment affects more than one-third of patients after an ischemic stroke (IS). Identifying markers of potential cognitive recovery after ischemic stroke can guide patients' selection for treatments, enrollment in clinical trials, and cognitive rehabilitation methods to restore cognitive abilities in post-stroke patients. Despite the burden of post-stroke cognitive impairment, biomarkers of cognitive recovery are an understudied area of research. This narrative review summarizes and critically reviews the current literature on the use and utility of neuroimaging as a predictive biomarker of cognitive recovery after IS. Most studies included in this review utilized structural Magnetic Resonance Imaging (MRI) to predict cognitive recovery after IS; these studies highlighted baseline markers of cerebral small vessel disease and cortical atrophy as predictors of cognitive recovery. Functional Magnetic Resonance Imaging (fMRI) using resting-state functional connectivity and Diffusion Imaging are potential biomarkers of cognitive recovery after IS, although more precise predictive tools are needed. Comparison of these studies is limited by heterogeneity in cognitive assessments. For all modalities, current findings need replication in larger samples. Although no neuroimaging tool is ready for use as a biomarker at this stage, these studies suggest a clinically meaningful role for neuroimaging in predicting post-stroke cognitive recovery. The American Heart Association (AHA) and the American Stroke Association (ASA) define ischemic stroke (IS) as an episode of neurological dysfunction caused by focal cerebral, spinal, or retinal infarction . IS reprn = 6,504, mean age = 73 years) found that one-third of patients cognitively improved during the first 3 months post-stroke, one-third deteriorated, and the rest remained cognitively unchanged  term “stroke” with the MeSH subheadings “complications” or “psychology” or the term “ischemic stroke” paired with both of the following terms in the abstract/title of each article or as MeSH terms:A cognition term .A neuroimaging term .Additional studies obtained through review of relevant article citations were included.Studies examining other types of strokes—hemorrhagic stroke (HS), traumatic stroke, subarachnoid hemorrhage (SH), and transient ischemic attacks (TIA)—were excluded. HS were excluded due to their different recovery trajectories compared to IS , 33. LikFinally, we required baseline imaging and cognitive assessments to be completed within 6 weeks of stroke. This is to ensure clinical relevance, as most imaging used to predict recovery would be completed during a hospital admission. A baseline cognitive assessment more than 6 weeks after an initial ischemic stroke may represent a different stage of stroke recovery and therefore not comparable to the other articles in this review.Studies of potential relevance were selected, and 35 were excluded after careful full-text review based on the criteria detailed above . A totalMRI as a means of characterizing or predicting cognitive impairment through structural biomarkers is well-documented , 36. TheAn important study by Sagnier et al. reported that first-time IS patients with pre-existing severe WMH on MRI had less improvement in verbal fluency tests at 3 months to 1 year after IS . SimilarSimilarly, Fruthwirth et al. found that deep WMH volume at baseline predicted recovery of set-shifting at 15 month follow up among patients with recent small subcortical infarcts: participants with no or mild deep WMH improved in set-shifting, while those with moderate to severe WMH showed no improvement . A similAnother study examining cerebral small vessel disease (mCSVD) score—determined by MRI evaluation—and medial temporal atrophy (MTA) score—a measure of hippocampal atrophy—found that neither of these imaging markers predicted change in MoCA scores between baseline and 1 year follow up . DespiteSagnier found that total gray matter (GM) volume was the only radiographic factor predictive of cognitive improvement at 12 month follow up among IS patients. Specifically, GM volume of left fronto-temporo-insular regions, right temporo-insular cortex, and basal ganglia were significantly associated with cognitive improvement .Several studies examined the impact of infarct location on cognitive recovery using structural MRI. Turunen examined differences in cognitive recovery for cortical vs. subcortical lesions among patients with first-ever supratentorial ischemic stroke . While tTogether, these studies support the use of structural MRI sequences to predict changes in cognition after ischemic stroke. Baseline measures of cerebral SVD, including WMH, as well as baseline cortical volume may be important indicators of the potential for restoration of cognitive ability after stroke. In addition, these articles suggest that different infarct locations as seen on structural MRI may be used to predict differing cognitive recovery trajectories after stroke, although more studies are needed to fully elucidate this relationship.Only one study reported post-IS cognitive recovery using resting-state fMRI (rs-fMRI)  40). Rs. Rs40). This study argues in favor of the potential of rsFC to predict the course of cognitive changes post-IS and the involvement of DMN in the recovery process, although more studies are required to confirm these findings and further understand brain functional networks involved in the acute to chronic phases of cognitive recovery . FurtherWhile fMRI examines the brain's functional integrity, DTI looks at the brain's white matter structural integrity. Fractional anisotropy (FA) is the most common parameter derived from DTI to assess brain structural connectivity. Other DTI measures are mean diffusivity (MD), radial diffusivity (RD), axial diffusivity (AD), independent of direction, and relative anisotropy.Important studies using DTI/DWI to study cognitive recovery after IS are summarized in In a different study using DWI, Kuceyeski et al.  comparedThe evidence for DTI and DWI as a means of predicting cognitive recovery after stroke is promising. More research is needed to determine the additional utility of this modality over structural MRI and to improve predictive value.To the best of our knowledge, this review is among the first to focus on neuroimaging biomarkers of cognitive recovery among IS patients. This review is limited by inherent challenges in using keywords to search literature, including a lack of consistent use of terminology to characterize study subject matter. We carefully reviewed the literature to ensure our search was as robust as possible but may have inadvertently missed relevant studies. One key aspect of our study that necessitated the exclusion of multiple otherwise relevant articles was that we focused specifically on the association of neuroimaging with cognitive recovery after stroke; each included study reported on the association between baseline neuroimaging and a change in a cognitive measure over time. Articles that merely reported a cognitive outcome  were therefore excluded from this review of neuroimaging biomarkers for cognitive recovery after IS.In addition, there are several gaps in the current literature, as discussed below:The best time to study cognitive recovery remains unclear and likely stems from uncertainty in the time frame of expected cognitive recovery post-stroke. In our review, we focused on baseline cognitive assessments done within 6 weeks of stroke to ensure clinical relevance and to adequately compare different articles. However, cognitive recovery studies among chronic stroke patients may help answer this question.The ability to detect cognitive recovery depends largely on the sensitivity of the cognitive test used. The studies included in this review used a variety of different tests—all outlined in To be included in this review, we required at least one repeat measure of cognition. However, it should be noted that having longitudinal imaging is also important to show concordance between neuroimaging and cognitive testing parameters across the recovery course.Some studies—which were excluded from this review—utilized cohorts of both ischemic (IS) and hemorrhagic strokes (HS) to study cognitive recovery. IS and IH have different pathophysiologies and different recovery processes , 33. AltOnly some of the reviewed studies explicitly assessed pre-stroke cognitive status. Pre-stroke cognitive status may influence the recovery process and should be taken into consideration . SimilarFinally, many studies in this review combined neuroimaging with non-neuroimaging tools to improve the prediction of cognitive recovery after IS. For example, Sangier created models that incorporated demographic and clinical factors, including age, sex, education, cardiovascular risk factors, and modified Rankin score , 43. SucIn summary, the current literature on cognitive recovery using neuroimaging as a predictive marker, although small, is promising. No imaging tool is ready for use as an established biomarker yet. Future studies should replicate current findings in larger samples using a consistent methodology.MT and VK conducted the literature search and drafted the manuscript in equal contribution. MP reviewed the manuscript for intellectual content. IN conceptualized the project and reviewed the manuscript for intellectual content.


Care management approaches are being widely tested in the Medicare-eligible population to manage chronic conditions, but few have focused on cognitive vulnerability as the pathway to optimizing independence in the community-dwelling older population. Cognitive vulnerability refers to living with dementia, depression, and/or a history of delirium. This presentation features a nurse practitioner-led team care management model (3D Team) to address cognitive vulnerability, tested in an ongoing clinical trial with older adults in a Medicare Advantage population. For older adults with dementia and their families served by the 3D Team, the nurse practitioner works closely with occupational therapists (OTs) delivering a nonpharmacological dementia care intervention. Preliminary results presented will include: characteristics of dyads that have received the dementia care intervention (N=70 dyads to date), how the nurse practitioner and OTs communicate, how the nurse practitioner reinforces dementia care skill-building strategies introduced by OTs, and process evaluation results to date.


Intratumour heterogeneity (ITH) has become an important focus of cancer research in recent years. ITH describes the cellular variation that enables tumour evolution, including tumour progression, metastasis and resistance to treatment. The selection and expansion of genetically distinct treatment-resistant cancer cell clones provides one explanation for treatment failure. However, tumour cell variation need not be genetically encoded. In pancreatic ductal adenocarcinoma (PDAC) in particular, the complex tumour microenvironment as well as crosstalk between tumour and stromal cells result in exceptionally variable tumour cell phenotypes that are also highly adaptable. In this review we discuss four different types of phenotypic heterogeneity within PDAC, from morphological to metabolic heterogeneity. We suggest that these different types of ITH are not independent, but, rather, can inform one another. Lastly, we highlight recent findings that suggest how therapeutic efforts may halt PDAC progression by constraining cellular heterogeneity. Pancreatic ductal adenocarcinoma (PDAC) is the most common and most aggressive type of pancreatic cancer with a five-year survival rate of only 8% . AlthougG12C mutation [G12C-mutated non-small cell lung cancer [The high proportion of metastatic disease at diagnosis is one of the main reasons for the high mortality rate of PDAC. The best curative treatment option is surgery, but even the small proportion of patients eligible for surgery has a five-year survival rate of only 30% . Both admutation , an inhig cancer .One reason for the poor chemotherapeutic response of PDAC tumours is their cellular heterogeneity. Whereas inter-tumour heterogeneity (differences between PDAC in different patients) can in principle be managed by careful classification of tumour subtypes and identification of subtype-specific vulnerabilities, intratumour heterogeneity (ITH) increases the likelihood that a subset of PDAC cells will be resistant to a given treatment, thwarting attempts at precision medicine. Genetic ITH, the existence of genetic subclones within the tumour of a single patient, was first demonstrated in PDAC by karyotyping  and lateIn this review, we discuss four types of ITH observed in PDAC—cancer “stemness”, transcriptional and epigenetic variation, the epithelial-to-mesenchymal spectrum, and metabolic differences. These phenotypic variables have all been associated with the aggressive and chemoresistant nature of PDAC. As we describe, these types of tumour cell heterogeneity are partly overlapping and often linked, although in many cases the triggers influencing the adoption of different cell states are not known. There is still much to discover about the impact of each variable on tumour progression, but recent studies have begun to dissect the drivers of heterogeneity itself, with the aim of reducing the complexity of PDAC to render it more clinically tractable.Like normal stem cells present in almost all organs, CSCs are defined by the two properties of self-renewal—the ability to give rise to more CSCs—and regenerative capacity—the ability to give rise to non-stem cells to recapitulate the histology of the tissue or tumour. In the case of PDAC, this usually results in a combination of epithelial and mesenchymal tumour cells, together with recruitment of stromal cells. CSCs therefore give rise to two types of ITH: cells differing in their tumour-initiating capacity (cancer stem versus non-stem cells) and cells arising from CSCs that differ in morphology and behaviour.The CSC model was first applied to leukaemia stem cells, with the term “cancer stem cell” coined in 2001 , and CSCTraditional conceptions of the CSC theory assume that cell-intrinsic mechanisms maintain a strict hierarchy of CSCs and non-CSCs . An impoG12D; Trp53fl/fl or Trp53fl/+; Pdx1-Cre) mouse model of PDAC. Although these mice eventually succumb to the disease, it is possible that Msi deletion is incomplete or compensated for by the other homologue. Even considering this caveat, Msi-deleted tumours show a more epithelial histology than their KPC counterparts [The mRNA-binding proteins Msi1 and Msi2 have been most comprehensively characterised in murine PDAC as CSC markers that also enhance tumour-initiating capacity. As an mRNA-binding protein, Msi2 directly binds and modulates the levels of mRNA molecules coding for epigenetic modifiers such as Brd4 and Hmga2 , explainterparts .high, but not CD9low, PDAC cells re-initiate tumour grafts that resemble the mixed—epithelial and mesenchymal—histology of primary KPC tumours. Mechanistically, CD9 interacts with the glutamine transporter ASCT2 and thus boosts glutamine import and downstream metabolism in CSCs. Heterozygous CD9 deletion in the KPC mouse model extends lifespan, suggesting that the facilitation of glutamine metabolism is an important function of CD9 in an autochthonous PDAC model. Several other studies also suggest that glutamine metabolism is critical to PDAC progression [More recently our laboratory identified the cell surface tetraspanin CD9 as a marker for both murine and human PDAC CSCs . CD9highgression –35, undeCross-species validation of CSC markers has sometimes yielded conflicting results. In mouse PDAC, some of the previously described human PDAC CSC markers including CD133, CD44, CD24 and ALDH1 activity fail to distinguish tumourigenic from non-tumourigenic cell populations . StudiesTaken together, the results from the studies described above point towards a CSC theory in PDAC in which there is no one fixed CSC subpopulation that is intrinsically hard-wired to act as a tumour-initiating population. Instead, there is a heterogeneously distributed CSC “state” within a given tumour , 37–39. + CSCs than primary tumours [An ideal anti-CSC therapy would prevent tumour cells from acquiring CSC capacity by steering all cells away from the stem-like state, but in practice, targeting existing CSCs via specific markers may be sufficient to reduce tumour heterogeneity and render PDAC more treatable. Given the data from a colorectal cancer model showing that metastases are more reliant on Lgr5 tumours  it will  tumours . In the Transcriptional subtypes of PDAC have proven considerably more robust than genetic driver mutations in classifying patients into relevant disease groups. Regulation of gene expression is multifaceted and may occur at both genetic and epigenetic levels. The epigenome—the myriad modifications that drive differences in gene expression independently of changes in DNA sequence—includes processes to silence or activate genes at the level of chromatin accessibility, histone modifications, DNA methylation and non-coding regulatory RNAs , 42. TheHowever, in many patient tumours, “squamous” morphology is observed heterogeneously within tumours, suggesting that these subtypes are not fixed but rather an emergent property of PDAC evolution. Intriguingly, tumours with subclonal mutations in chromatin modifiers such as KDM6A and ARID1A are more likely to exhibit histological features of the squamous subtype, suggesting this broad family of mutations predispose PDAC cells to adopt poor prognostic features . Figure G12D; Trp53R172H/+; Pdx1-Cre) organoids implicates broad enhancer reprogramming as a mechanism imbuing PDAC cells with metastatic competence [One area in which epigenetic heterogeneity has been unequivocally demonstrated within individual patients is when comparing primary tumours and their metastases. Studies of paired PDAC samples from primary tumours and metastases, in both mouse and human rapid-autopsy specimens, have identified broad epigenetic reprogramming and changes in chromatin modifications strongly associated with metastasis. Analyses of the chromatin landscape of paired metastasis-derived and primary tumour-derived KP*C  is a dynamic, reversible process in which epithelial cells lose their local attachments and apical-basal polarity to adopt a spindle-shaped morphology and increased motility . This moG12D-mutant pancreatic epithelial cells spontaneously escape replicative senescence to generate two distinct tumour cell phenotypes—one epithelial and the other mesenchymal—each with distinct molecular drivers for survival and proliferation [Intense research interest in EMT has been driven in part due to its association with invasion and metastasis. In the KPC model, mesenchymal-like cells can be identified in the earliest stages of pancreatic cancer, even preceding the formation of a defined primary tumour . These mferation . MesenchThe significance of EMT in PDAC and other solid tumours in humans has been historically more controversial . NonetheEMT programmes are orchestrated by a group of transcription factors, the EMT-TFs, of which TWIST1, ZEB1/2 and SNAI1/2 are the most comprehensively studied , though Snail and Slug. Deletion of Grem1 leads to an almost complete switch of cancer cells to a mesenchymal phenotype, with an associated increase in metastatic frequency. Thus, a single soluble factor, GREM1, ensures the co-existence of both epithelial and mesenchymal PDAC subpopulations [We recently demonstrated that an interaction between the epithelial and mesenchymal subpopulations regulates EMT in PDAC. We found that the BMP inhibitor GREM1 is highly expressed in mesenchymal subpopulations of KPC tumours and acts in a paracrine manner to suppress EMT in the epithelial subpopulation, by reducing expression of the EMT-TFs ulations .More nuanced prospective isolation strategies and the application of single-cell technologies have further complicated the classical binary model of EMT. Recent evidence, from multiple tissue and tumour types, supports “partial EMT” states, which exhibit transcriptional characteristics of both epithelial and mesenchymal cells . These pNonetheless, there are several unresolved questions related to EMT and PDAC. The majority of PDAC metastases in both genetically engineered mouse models and human patients have an epithelial histology , 89, andThe fact that tumour metabolism differs from that of its organ of origin was first recognised almost a century ago by Otto Warburg. Solid tumours are generally more glycolytic than healthy tissues even when oxygen is not limiting . This aeConstitutively active mutant KRAS, which drives over 90% of all PDAC cases , is a maISL2 is epigenetically silenced in only some regions of human PDAC tumours, and ISL2 downregulation leads to enhanced expression of genes involved in oxidative phosphorylation, thus potentially rendering those cells sensitive to mitochondrial electron transport chain inhibitors [Intratumour metabolic heterogeneity is much more challenging to study for technical reasons, but given that differences in metabolic pathways have been described as potential drivers of the CSC phenotype in PDAC , 25, it hibitors .Several studies have reported differences in metabolism between different CSC-enriched populations and the bulk of PDAC cells. Human PDAC cells grown as tumour spheres are less glycolytic than cells grown in adherent culture, relying more on oxidative phosphorylation, and are more sensitive to the mitochondrial electron transport chain inhibitor metformin. Tumour spheres are also better able to tolerate low glucose or low glutamine conditions . HoweverSeveral findings highlight the metabolic plasticity of PDAC cells upon perturbation. As an example, whereas heterozygous CD9 knockout prolonged survival in KPC mice, homozygous CD9 knockout did not. Metabolomic profiling showed that homozygous CD9 knockout cells were more similar to CD9 wild-type cells than to heterozygous knockouts, indicating that homozygous knockout leads to compensatory metabolic rewiring to maintain high levels of glutamine metabolism . SimilarA common product of PDAC metabolism is lactate, which needs to be exported from cells, for example via monocarboxylate transporters (MCTs), to maintain intracellular pH in a physiological range. High levels of MCT4 mark highly aggressive, glycolytic tumours and human PDAC cell lines. However, MCT4 knockdown leads to upregulation of an alternative transporter, MCT1, as well as to compensatory mitochondrial oxidative phosphorylation and autophagy . In the SMAD4 is deleted in approximately 30% of PDAC samples [SMAD4 loss correlating with higher levels of PGK1 in patient samples and upregulation of PGK1 in human PDAC cell lines. Of note, PGK1 expression is heterogeneous across human PDAC samples, with both the total levels of the protein as well as its intracellular localisation varying, suggesting that within SMAD4-negative tumours there are differences in metabolic states. Higher levels of cytoplasmic PGK1 stimulate glycolysis, but PGK1 can also translocate into the nucleus to act as a transcriptional repressor of CDH1  [The gene encoding the tumour suppressor  samples . A recenadherin) . How thiadherin)  remains adherin) .Solid tumours contain both normoxic as well as hypoxic regions depending on their proximity to intact blood vessels. These hypoxic regions are particularly pronounced in PDAC due to the extensive desmoplastic stromal reaction that surrounds pockets of tumour cells . FurtherHigh rates of lactate production and extrusion into the extracellular environment can lead to chronic acidosis, which in turn leads to oxidative stress . To combPDAC has proven remarkably recalcitrant to strategies targeting global tumour cell proliferation, making it critical to dissect the drivers of tumour heterogeneity. Despite considerable progress in this area, ITH remains a significant challenge in pancreatic cancer and likely contributes to the high morbidity and mortality of the disease. Unlike many other tumour types, which acquire new subclonal driver mutations during tumour evolution, PDAC frequently has multiple clonal driver mutations at its root, with comparably low genetic heterogeneity within the tumour . InsteadThe recent explosion of experimentally tractable human primary cell systems, such as patient-derived organoids , 131 andOverall, a better understanding of ITH may lead to better treatment options for patients, either through combinatorial approaches targeting multiple aspects of tumour biology, or by reducing the tumour heterogeneity itself in order to increase chemoresponsiveness. As tools become available to rapidly discover, test and translate new therapeutic strategies, the prospect of truly durable clinical responses may become a reality.


Concerns about health and fitness during lockdown may serve as a trigger for eating disorders in vulnerable individuals. Other risk factors may also include increased use of social networks and comparison with beauty ideals. Isolation, loneliness and problems with emotional regulation may lead people to reduce food intake by giving them a greater sense of control.Emphasise the relevance of the increase in the incidence of Eating Disorders (ED) cases during the pandemic.Review of the scientific literature based on a relevant clinical case.14-year-old female, residing with her mother. She reports that from the beginning of COVID-19 confinement she became obsessed with leading a healthier life, starting to restrict food, limiting fats and carbohydrates, and having also started compulsive physical activity (approximately 4 hours of aerobic exercise per day), without associated purging behaviours. She also acknowledges eating small amounts  and controlling all calories, stating that food and practices aimed at “staying healthy” now dominate her life. Her previous BMI was 18, with a current BMI of 11.7.Patients suffering from ED, who often have poor knowledge of their illness and find social-emotional communication difficult, may delay seeking help. Studies suggest the relevance of identifying specific vulnerability factors among ED patients in confinement in order to develop preventive strategies and personalised treatment approaches.No significant relationships.


Smoking among cancer survivors can increase the risk of cancer reoccurrence, reduce treatment effectiveness and decrease quality of life. Cancer survivors without health insurance have higher rates of smoking and decreased probability of quitting smoking than cancer survivors with health insurance. This study examines the associations of the Affordable Care Act (ACA) Medicaid insurance expansion with smoking cessation assistance and quitting smoking among cancer survivors seen in community health centers (CHCs).Using electronic health record data from 337 primary care community health centers in 12 states that expanded Medicaid eligibility and 273 CHCs in 8 states that did not expand, we identified adult cancer survivors with a smoking status indicating current smoking within 6 months prior to ACA expansion in 2014 and ≥ 1 visit with smoking status assessed within 24-months post-expansion. Using an observational cohort propensity score weighted approach and logistic generalized estimating equation regression, we compared odds of quitting smoking, having a cessation medication ordered, and having ≥6 visits within the post-expansion period among cancer survivors in Medicaid expansion versus non-expansion states.Cancer survivors in expansion states had higher odds of having a smoking cessation medication order  and higher odds of having ≥6 office visits than those in non-expansion states . Odds of quitting smoking did not differ significantly between patients in Medicaid expansion versus non-expansion states.The increased odds of having a smoking cessation medication order among cancer survivors seen in Medicaid expansion states compared with those seen in non-expansion states provides evidence of the importance of health insurance coverage in accessing evidence-based tobacco treatment within CHCs. Continued research is needed to understand why, despite increased odds of having a cessation medication prescribed, odds of quitting smoking were not significantly higher among cancer survivors in Medicaid expansion states compared to non-expansion states. Approximately 30% of cancer deaths in the United States (US) are caused by tobacco use and smoking . SmokingA study using data from the 2017 National Health Interview Survey (NHIS) found 13% of adult cancer survivors reported current smoking  and apprCancer survivors without health insurance have higher rates of smoking and decreased probability of quitting smoking than cancer survivors with insurance –10. A stThe Patient Protection and Affordable Care Act (ACA) mandate called for the expansion of Medicaid coverage  to all adults earning less than or equal to 138% of the federal poverty level (FPL). Following a Supreme Court ruling, states were allowed to choose whether or not to expand Medicaid. As of February 2022, 39 states had adopted the Medicaid expansion and 12 states had not [The ACA mandate also required that insurers cover certain preventive services, including smoking cessation interventions, with no cost sharing for newly eligible Medicaid beneficiaries in states that expanded Medicaid . This prTherefore, this retrospective observational cohort study sought to examine the odds of smoking cessation assistance and cessation among cancer survivors who were patients of community health centers (CHCs) in states that expanded Medicaid via the ACA compared to those in non-expansion states. CHC settings are important to study given that they provide primary care services, including smoking cessation services, to many uninsured or Medicaid-insured patients with higher rates of smoking than the general population , 23. NatWe used electronic health record (EHR) data from the Accelerating Data Value Across a National Community Health Center Network (ADVANCE) Clinical Research Network (CRN) , a membeIn this observational cohort study, we included 337 primary care CHCs across 12 states that expanded Medicaid eligibility to ≤138% of the FPL for all adults including those without dependent children as of 1/1/2014  and 273 CHCs in 8 non-expansion states . We included Wisconsin as an expansion state because it opened Medicaid coverage to adults earning up to 100% of the FPL (near the threshold of ≤138% of the FPL). We included Alaska, Indiana, and Montana as non-expansion states because they did not expand Medicaid until late in our post-ACA study period . Our designation of states as expansion vs. non-expansion are similar to other studies assessing the impact of the ACA on health care outcomes and utilization , 27–29.We assessed patients 12 months pre-ACA (1/1/2013-12/31/2013) and 24 months post-ACA Medicaid expansion (1/1/2014-12/31/2015). We identified cancer survivors through their medical histories, encounter diagnoses, and problem-list records up to the date of their last pre-ACA visit. We included cancer survivors aged 19-64 throughout the entire study period, who had ≥1 pre-ACA visit to a study CHC and whose last recorded tobacco use status during this time period indicated current smoking , and who had ≥1 post-ACA visit with a documented smoking assessment. Based on the US National Cancer Institute’s definition, we consider a cancer survivor to be anyone alive who has ever had a cancer diagnosis regardless of where they are in the course of their disease . We exclOutcomes included quitting smoking (≥1 report of quitting), provision of smoking cessation medication (≥1 prescription of a cessation medication) and utilization of primary care (≥6 vs. < 6 visits) in the 24-month post-period. The EHR presents a discrete data field for smoking status at each primary care encounter, which can be confirmed, updated, or not reviewed. Smoking cessation (‘quit’) at ≥1 visit during the post-period was coded as a binary yes/no variable. Using methods similar to prior EHR-based studies –35, a peOur independent variable was Medicaid expansion status: patients from CHCs in states that expanded versus did not expand.To describe patients living in expansion and non-expansion states and to develop weights to control for differences in expansion groups, we used the following, EHR-derived pre-ACA covariates: sex, age as of 1/1/2014, race/ethnicity , household income as percent of FPL as of 1/1/2014, location of patient’s primary clinic , number of ambulatory visits in 2013, insurance status at visits in 2013 , and the Charlson Comorbidity Index . We exclTo analyze whether cancer survivors in expansion states who reported current smoking prior to the ACA expansion would have better smoking cessation outcomes than those in non-expansion states, we used a propensity score (PS) weighted approach.We calculated inverse probability of treatment weights (IPTW) to control for differences in pre-ACA patient-level characteristics between the expansion and non-expansion groups. In this approach, patients are weighted by the inverse of the probability of being sampled from the treatment  group. We first fit a logistic regression model for expansion status including all patient characteristics described above as covariates, to obtain each patient’s PS or probability of being in an expansion state. We then calculated stabilized IPTWs to create a pseudo-population close to our original sample size. To assess balance before and after weighting, we computed standardized differences, as they are not unduly influenced by sample sizes. We considered covariates with absolute standardized mean differences (ASMD) ≤0.1 in the weighted sample to be free of residual imbalance that would influence final models. We calculated effective sample size, which was the approximate number of observations under simple random sampling that would produce a variation equivalent to that of the IPTW sample.Using the PS-weighted sample, we computed adjusted odds ratios (aOR) and predicted marginal probabilities of quitting smoking, having a cessation medication order, or having ≥6 ambulatory visits in the post-period. We used generalized estimating equation (GEE) logistic regression models to account for clustering of patients within CHCs. To account for potential differences in data handling, we included EHR network (i.e. OCHIN vs. HCN) as the only covariate in our GEE models. All GEE models assumed an exchangeable correlation structure and applied a robust sandwich variance estimator to account for possible misspecification . All anaTable Figure The ACA expansion resulted in health insurance coverage for millions of US adults not previously Medicaid-eligible . In the Despite these increased odds of smoking cessation medication orders among cancer survivors in expansion versus non-expansion states, we did not observe statistically significant differences in odds of quitting. These findings are in contrast to those of a previous study that found higher odds of quitting smoking among adult patients of CHCs in expansion states compared with those in non-expansion states; however, this prior study included the entire patient population and subanalyses for cancer survivors were not performed . We postIt also is possible that current smoking cessation interventions, including pharmacotherapy, may not be as effective among cancer survivors compared to patients without a history of cancer. One meta-analysis of randomized controlled trials designed to promote smoking cessation among cancer survivors supports this conclusion; however, the quality of the included interventions was mixed and the authors caution in drawing firm conclusions based on the present evidence . It alsoThe importance of CHCs for smoking cessation assistance among cancer survivors should also be highlighted. In the overall study sample, 30% of cancer survivors received smoking cessation medications from their CHC. The American College of Surgeons emphasizes that providing a high level of quality care to cancer survivors requires coordination of care among many medical disciplines, including primary care providers . Much reWe had follow-up smoking status for patients who had a return clinic visit, and therefore, cannot determine the quit status of patients who did not return for various reasons . That said, a previous study found that about 80% of CHC patients with a chronic health problem return for ≥1 visit within a three-year period . We alsoCancer survivors in ACA Medicaid expansion states had more than twice the odds of having a smoking cessation medication prescribed compared with those from non-expansion states, providing evidence of the importance of insurance coverage in accessing evidence-based tobacco treatment within the CHC setting. Our study findings support the need for continued efforts to ensure health insurance coverage for primary care-based tobacco treatment for socioeconomically disadvantaged cancer survivors. Further, research is needed to understand why, despite increased odds of having a cessation medication prescribed, odds of quitting were not significantly higher among cancer survivors in Medicaid expansion states. If our findings are replicated, interventions tailored to the specific needs of cancer survivors might be warranted.