FABP1 levels were correlated with radiographic and blood-based markers of congestion, hemodynamic derangements during peak exercise , reduced exercise cardiac output (r = − 0.49), and poor exercise workload achieved . FABP1 distinguished HFpEF from controls with an area under the curve of 0.79 (p = 0.003) and had an incremental diagnostic value over the H2FPEF score (p = 0.007). In conclusion, FABP1 could be a novel hepatic biomarker that associates with hemodynamic derangements, reduced cardiac output, and poor exercise capacity in HFpEF.Elevated intracardiac pressure at rest and/or exercise is a fundamental abnormality in heart failure with preserved ejection fraction (HFpEF). Fatty acid-binding protein 1 (FABP1) is proposed to be a sensitive biomarker for liver injury. We sought to determine whether FABP1 at rest would be elevated in HFpEF and would correlate with echocardiographic markers of intracardiac pressures at rest and during exercise. In this prospective study, subjects with HFpEF (n = 22) and control subjects without HF (n = 23) underwent resting FABP1 measurements and supine bicycle exercise echocardiography. Although levels of conventional hepatic enzymes were similar between groups, FABP1 levels were elevated in HFpEF compared to controls (45 [25–68] vs. 18 [14–24] ng/mL,  Increases in cardiac filling pressures are a fundamental hemodynamic abnormality in patients with HFpEF and high filling pressures are associated with symptoms of dyspnea, poor aerobic capacity, and worse clinical outcomes3. In patients with HFpEF, left ventricular (LV) filling pressures are often normal at rest, but become abnormally increased only during physiological stress such as exercise5. Recent studies have demonstrated the importance of exercise stress testing such as diastolic stress echocardiography to unmask hemodynamic abnormalities during exercise and to enhance the diagnosis of HFpEF10.Approximately half of the patients with heart failure (HF) have a preserved ejection fraction (HFpEF)12. A number of other candidate biomarkers that may relate to the pathophysiology of HFpEF have been reported, including myocardial injury and stress, neurohormonal activation, inflammation, and metabolism16. However, very little data are available regarding the biomarkers representing the cardio-hepatic interactions. Fatty acid-binding protein 1 (FABP1), also known as liver-FABP, is 14.4-kDa protein, which is primarily expressed in the liver18. Circulating FABP1 levels are induced by liver injuries such as acute and chronic hepatitis, liver cirrhosis, and nonalcoholic steatohepatitis21. Emerging data have suggested that FABP1 is a sensitive marker to detect early liver injury22. Our group demonstrated that FABP1 levels were elevated in HF patients compared to controls and predicted poor clinical outcomes23.Natriuretic peptides are the most commonly used biomarker to help the diagnosis of HFpEF and are endorsed in consensus guidelines to facilitate the diagnosis of HFpEF2FPEF score.Given this background, we hypothesized that FABP1 levels at rest would be elevated in patients with HFpEF compared to control subjects free of HF and that the magnitude of elevation would correlate with hemodynamic perturbations at rest and during exercise in HFpEF. We also hypothesized that FABP1 would have an incremental diagnostic value to identify HFpEF over the validated H2FPEF score was higher in patients with HFpEF than controls. Hemoglobin and creatinine levels were similar between HFpEF patients and controls.We enrolled 23 control subjects and 22 HFpEF patients in this study. Compared to control subjects, patients with HFpEF were older and had radiographic and blood-based signs of congestion, evidenced by a higher prevalence of cardiomegaly and greater N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels Table . Sex, bop = 0.04) or any hepatobiliary enzymes . FABP1 levels were not correlated with conventional hepatobiliary enzymes . In contrast, FABP1 levels were directly correlated with radiographic and blood biomarkers of congestion  and PA systolic pressure (PASP) while there were marginal correlations with E/e’ ratio and RA pressure  , mPAP , PASP , and RAP  although its circulating levels were similar in HFpEF and controls (p = 0.14).At rest, heart rate, systolic blood pressure (BP), and oxygen saturation were similar between HFpEF and control subjects Table . Comparep = 0.007 and r = − 0.34, p = 0.03). Resting FABP1 levels were also correlated with poorer LV diastolic reserve , higher LV filling pressure , more severe pulmonary hypertension and RA hypertension , and lower CO  during peak exercise . Modest correlations were observed between other hepatobiliary markers and echocardiographic parameters during peak exercise among all participants  and peak exercise, heart rate, systolic BP, and oxygen saturation were similar between groups Table . Compare0 W and p2FPEF score and NT-proBNP levels demonstrated good discriminatory abilities for identifying HFpEF, with areas under the curve (AUCs) of 0.72 and 0.88 (p = 0.009 and p < 0.0001), respectively. FABP1 distinguished HFpEF from control subjects with an AUC of 0.79 (p = 0.003) whereas other hepatobiliary markers did not .As expected, The Hot Table . FABP1 hp = 0.0009); (2) FABP1 remained significantly associated with NT-proBNP levels , exercise E/e’ , exercise eRAP , exercise PASP , exercise CO , and exercise workload achieved ; and (3) FABP1 could distinguish HFpEF from controls with an AUC of 0.80 (p = 0.002), with an incremental diagnostic value over the H2FPEF score .Of the 45 participants, liver sonographic examinations that were performed within a year from exercise echocardiography were available in six patients. Of the six patients, two patients were found to have mild chronic hepatitis and one had mild fatty liver while no evidence of acute or chronic hepatitis was observed in the others. Sensitivity analyses were then performed excluding the three patients with liver diseases. We found that (1) FABP1 levels remained significantly higher in HFpEF patients than controls  abundantly expressed in a tissue-restricted manner; therefore, in response to tissue injury, FABPs diffuse more rapidly through the interstitial space and the endothelial clefts to circulation than large proteins such as alanine transaminase (ALT) (96 kDa) or AST (90 kDa)25. As such, FABP1 could serve as a biomarker for an earlier phase of hepatic injury where conventional liver markers are not released. In keeping with this notion, our data showed that circulating FABP1 levels were increased in the absence of elevation of AST and ALT. Accordingly, one of the most likely mechanisms for FABP1 elevation in HFpEF patients is a response to early or minimal hepatic injury.Biomarkers provide valuable information to understand the specific pathophysiological pathways that relate to the diseaseHowever, our simple correlation analyses revealed no significant correlation between FABP1 and AST, ALT, or the other conventional hepatic enzymes, arguing against the hepatic injury as a sole mechanism of an increase in circulating FABP1 in HFpEF. We recently found that FABP1 levels were significantly increased during exercise and were significantly correlated with plasma norepinephrine levels in healthy volunteers (manuscript in preparation). These results suggest that exercise induces circulating FABP1 through the mechanisms involving sympathetic nervous system activation. Thus, it is intriguing to speculate that an elevation of FABP1 in HFpEF is partly due to a hepatic activation of adrenergic signaling. Further studies are needed to determine the mechanisms underlying elevation in FABP1 in patients with HFpEF.26. The current study demonstrated correlations of FABP1 levels with radiographic and blood markers of congestion and echocardiographic evidence of hemodynamic derangements . The cross-sectional design of our study cannot determine whether hemodynamic derangements caused hepatic injury to promote elevations in circulating FABP1 levels, or whether FABP1 directly worsened LV diastolic function and hemodynamics during exercise. It has been shown that FABP1 is an effective endogenous cytoprotectant, minimizing hepatocyte oxidative damage27. The elevation of circulating FABP1 may represent a compensatory mechanism to counteract oxidative stress and inflammation in the liver21. Based on these data and ours, we speculate that systemic venous congestion secondary to the elevation in right heart pressures may lead to hepatocyte injury to promote up-regulation of FABP1. Further studies are warranted to determine the mechanisms underlying hepatic injury in HFpEF.HFpEF is a clinical syndrome that can be characterized by reduced cardiovascular reserve which leads to an elevation in LV filling pressure and secondary pulmonary hypertension during exercise10. Assessments of clinical characteristics, chest radiography, echocardiography, and blood biomarkers play an important role in the diagnostic evaluation of HFpEF. Natriuretic peptides are the most commonly-used blood-based biomarker to facilitate diagnosis of HFpEF and a recent guideline statement from the ESC has proposed a scoring system based upon echocardiographic markers of diastolic function as well as natriuretic peptides to determine whether HFpEF is present7. However, there are well-known limitations of natriuretic peptides, such as an underestimation in obese patients30. This makes the identification of novel biomarkers that relate to greater elevations of cardiac filling pressures or the presence of HFpEF high priority. It is therefore noteworthy that FABP1 levels were elevated in patients with HFpEF compared to controls, were well correlated with echocardiographic markers of elevated cardiac filling pressures and PA pressures during exercise, and predicted the presence of HFpEF. Although the diagnostic ability of FABP1 was lower than NT-proBNP, FABP1 had an incremental value to identify HFpEF from control subjects over the established H2FPEF score. The current data suggest that FABP1 could be a candidate biomarker to help identify HFpEF among patients with chronic dyspnea. Further large-scale studies are required to validate these findings and establish a cut-off for FABP1 levels to allow for incorporation into current diagnostic practice.Diagnosis of HFpEF in people presenting with chronic dyspnea is challenging23. In other words, the previous study was a longitudinal outcome study in design. On the other hand, the present study was a cross-sectional study to investigate whether FABP1 levels would correlate with echocardiographic markers of intracardiac pressures during supine bicycle exercise. Regarding the population, the previous study included HF patients regardless of EF and control subjects who were referred to coronary angiography. On the other hand, the present study included HFpEF patients and control subjects who were referred to exercise stress echocardiography for the evaluation of unexplained dyspnea. The pathophysiologic role of FABP1 in HF with reduced EF was beyond our scope. Patients with liver disorders were excluded from the analysis based on liver enzymes. Liver sonographic data were available in six patients, in which two had mild chronic hepatitis and one had mild fatty liver. We cannot exclude the possibility that some patients who did not undergo liver sonography might have had a liver disease that was not evident from liver enzymes31. The presence of hepatitis could have biased the results although key results remained similar even after excluding the three patients. The control group was not normal as they were referred for exercise stress echocardiography in the evaluation of exertional dyspnea and had comorbidities such as hypertension and interstitial pneumonia and relatively higher FABP1 levels than healthy controls, which could also bias the results22. However, the fact that the control population was more diseased than a truly normal healthy control population only biases our data toward the null. Given the presence of exertional dyspnea and comorbidity burden, control subjects might be considered as pre-HFpEF, and the inclusion of controls might add greater insight into the continuous relationships between the magnitude of FABP1 elevations and cardiac abnormalities across the spectrum from risk to frank HFpEF. We cannot conclude that these observations were specific to HFpEF, or may be observed in other disorders that cause right heart pressures, such as HF with reduced EF or non-Group II pulmonary artery hypertension. Further studies are required to address this question. The small sample size of this study does not allow to simultaneously adjust multivariable factors to analyze the diagnostic power of FABP1 to distinguish HFpEF from controls.This is a single-center study from a tertiary referral center. All participants were referred for exercise stress echocardiography for the evaluation of unexplained exertional dyspnea, introducing selection and referral bias. Although the current study and our previous one both focused on FABP1 levels in HF, the two studies are essentially different in three main perspectives: the aim, study design, and population. The primary aim of the previous study was to determine the prognostic value of FABP1 in HFSerum FABP1 levels are elevated in early HFpEF and the magnitude of elevation is associated with echocardiographic markers of elevated LV filling pressure and PA pressures, systemic congestion, and lower workload. The present study suggests that FABP1 may serve as a potential hepatic biomarker that associates with hemodynamic perturbation, lower cardiac output, and reduced exercise capacity in HFpEF, and that FABP1 may help distinguish HFpEF among subjects with dyspnea. Further studies are required to confirm the current findings.32. Subjects who were referred to exercise echocardiography were also screened as a comparator group (non-cardiac dyspnea: controls). Control subjects were required to have (1) exertional dyspnea and (2) no objective evidence of elevated left heart filling pressures at rest and with exercise (criteria above). Subjects with EF < 50%, significant left-sided valvular heart disease , infiltrative, restrictive, or hypertrophic cardiomyopathy, non-Group II pulmonary artery hypertension or exercise-induced pulmonary hypertension without elevation of E/e’ 34, and significant liver disorders  were excluded. There was no overlap with our previous study focusing on the prognostic value of FABP1 in HF patients regarding the study subjects23. The data underlying this article will be shared on reasonable request to the corresponding author.This was a cross-sectional study that assessed the association between serum FABP1 levels and Doppler echocardiographic hemodynamics at rest and during supine bicycle exercise. We prospectively enrolled consecutive subjects who were referred to our echocardiographic laboratory for exercise stress echocardiography for the evaluation of unexplained exertional dyspnea between November 2019 and June 2020. The study was approved by our Institutional Review Board  and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained by all participants. HFpEF was defined by typical clinical symptoms , normal LV EF (≥ 50%), and objective evidence of elevated left heart filling pressures at rest and/or with exercise: (1) the European Society of Cardiology (ESC) and American Society of Echocardiography/European Association of Cardiovascular Imaging (ASE/EACVI)-recommended echocardiographic diastolic dysfunction; (2) the E/e’ during exercise > 15; or (3) invasively-measured pulmonary capillary wedge pressure [PCWP] at rest > 15 mmHg and/or with supine ergometry exercise ≥ 25mmHgVenous blood samples were obtained just before the assessment of exercise stress echocardiography. Serum FABP1 levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit . As specified by the manufacturer, the lower limits of detection of serum FABP1 were 9.4 pg/mL. Serum NT-proBNP levels were also determined using another ELISA kit . Serum hemoglobin, hepatobiliary enzymes, creatinine, glucose, and lipid profiles were measured by routine automated laboratory procedures.35. LV systolic function was assessed based on the EF and the systolic mitral annular tissue velocity at the septal annulus . LV diastolic function was assessed using the E, e’, and the E/e’ ratio. Left atrial volume was determined using the biplane method of disks. Stroke volume (SV) was determined from the LV outflow dimension and pulse wave Doppler profile. CO was calculated from the product of heart rate and SV. RV systolic function was assessed using TAPSE and TV s’. RA pressure was estimated from the diameter of the inferior vena cava and its respiratory change. PASP was calculated as 4 × (peak tricuspid regurgitation [TR] velocity)2 + estimated RA pressure. The mPAP was calculated as 0.61 × PASP + 236.Comprehensive resting echocardiography was performed by experienced sonographers using a commercially available ultrasound system . LV volumes and EF were determined using apical 4-chamber viewsSubjects underwent supine cycle ergometry echocardiography, starting at 20 W for five minutes, increasing 20 W increments in three-minute stages to subject-reported exhaustion. Echocardiographic images were obtained at baseline and during all stages of exercise. All Doppler measures represent the mean of ≥ three beats. All studies were interpreted offline and in a blinded fashion by a single investigator (M.O.).2FPEF score is an evidence-based algorithm that incorporates echocardiographic and clinical variables, which has been shown to provide a valid estimate for HFpEF probability6. The H2FPEF score is based on four clinical parameters  and two echocardiographic variables (E/e’ ratio > 9 [1 point] and PASP > 35 mmHg [1 point]). This results in a categorical H2FPEF score ranging from 0 to 9.The H2FPEF score was evaluated by sequential logistic regression analysis using nested models37. The change in overall − 2 log likelihood ratios of the models was used to assess the increment in diagnostic information. All tests were two-sided, with a value of p < 0.05 considered significant. All statistical analyses were performed with JMP 14.0.0 .Data are reported as mean (SD), median (IQR), or number (%) unless otherwise specified. Between-group differences were compared by unpaired t-test, Wilcoxon rank-sum test, or chi-square test, as appropriate. Pearson’s or Spearman’s correlation coefficients were used to assess relationships between two variables of interest, where non-normally distributed data were log-transformed. Multivariable linear regression analysis was used to test whether differences in FABP1 remain significant after adjusting confounding factors. Receiver operating curves (ROC) were constructed to evaluate the diagnostic ability of FABP1. The incremental diagnostic value of FABP1 to the H


Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome responsible for high mortality and morbidity rates. It has an ever growing social and economic impact and a deeper knowledge of molecular and pathophysiological basis is essential for the ideal management of HFpEF patients. The association between HFpEF and traditional cardiovascular risk factors is known. However, myocardial alterations, as well as pathophysiological mechanisms involved are not completely defined. Under the definition of HFpEF there is a wide spectrum of different myocardial structural alterations. Myocardial hypertrophy and fibrosis, coronary microvascular dysfunction, oxidative stress and inflammation are only some of the main pathological detectable processes. Furthermore, there is a lack of effective pharmacological targets to improve HFpEF patients’ outcomes and risk factors control is the primary and unique approach to treat those patients. Myocardial tissue characterization, through invasive and non-invasive techniques, such as endomyocardial biopsy and cardiac magnetic resonance respectively, may represent the starting point to understand the genetic, molecular and pathophysiological mechanisms underlying this complex syndrome. The correlation between histopathological findings and imaging aspects may be the future challenge for the earlier and large-scale HFpEF diagnosis, in order to plan a specific and effective treatment able to modify the disease’s natural course. Heart failure with preserved ejection fraction (HFpEF) is a complex and multifaceted clinical syndrome. HFpEF percentage is between 22% and 73% of all heart failure (HF) cases ,2,3,4 anIn HFpEF, the underlying myocardial alterations are incompletely defined. Myocardial histopathological alterations have been characterized through invasive methods, such as endomyocardial biopsy, but also cardiac magnetic resonance (CMR) or other imaging technique are able to identify them in a simpler and non-invasive way. However, the correlation between histopathological findings and imaging aspects is still a challenge for medicine. Myocardial fibrosis, cardiomyocytes hypertrophy, coronary microvascular dysfunction (CMD) and inflammation have been identified as major pathological processes that affect HFpEF myocardium. Although these mechanisms have been also identified in HFrEF, several observations highlight that their spatial and temporal onset and progression may differ between the two conditions. HFpEF hearts appear remodeled towards a hypertrophic shape, and not dilated as HFrEF, left atrial dilation is very frequent and most of HFpEF patients have evidence of diastolic dysfunction. In this regard, the advanced imaging techniques, and in particular the refined tissue characterization allowed by CMR is a very valid tool . For an Myocardial tissue characterization is a crucial aspect for the comprehension of HFpEF molecular basis, as well as pathophysiological aspects. Currently, there is no specific therapy for HFpEF and risk factors control is the primary and unique approach for HFpEF management and prevention . T1 MappThe aim of this review is to provide an overview about the most recent findings about myocardial tissue characterization in HFpEF, through invasive and non-invasive methods, in order to allow a wide comprehension of HFpEF pathophysiological mechanisms and provide new therapeutic targets for its treatment. HF is a complex syndrome characterized by the inability of heart to guarantee an adequate blood output to peripheral organs or to provide it through cardiac filling pressure increase. The definition of HFpEF is clinical and coupled with the evidence of a left ventricular ejection fraction >50% .Pathophysiology of HFpEF is less deciphered than HFrEF, since there are different underlying etiologies, pathways, phenotypes and comorbidities. From the pathophysiological point of view, in HFpEF, left ventricular concentric remodeling and/or hypertrophy have been observed ,7,8. HFpMyocardial fibrosis is characterized by extracellular matrix components storage. Myocardial fibrosis is a specific histopathological process related to myocardial remodeling and it is closely associated with HF. This histopathological mechanism underpins many genetic, epigenetic and molecular abnormalities, such as excessive extracellular matrix production, post-translational collagen cross linking abnormalities, as well as matrix metalloproteinases dysregulation ,8,37. Mylocus minoris resistentiae developed in the infarct area. On the other hand, reactive fibrosis is more associated with non-ischemic myocardial involvement, in condition of pressure overload [From the clinical point of view, the myocardial fibrosis spatial distribution and temporal onset may play a prognostic role, being involved in systolic and diastolic dysfunction, as well as arrythmias ,40,41,42overload . Specifioverload ,44,45. Roverload . Both fooverload  Specificoverload . Not onloverload ,48. Su eoverload . HoweverConsidering the hemodynamic and clinical impact of myocardial fibrosis, several aspects should be related with the increasing left ventricular filling pressure, worse diastolic function and hospitalization, in HFpEF patients. In this regard, type 1 collagen production instead of type III collagen, a raise in collagen storage and cross linking have been observed ,49,50,51Cardiac amyloidosis is one of the causes for HFpEF and its prevalence, in this population, is constantly increasing . TransthReduced left ventricular compliance and relaxation are mainly due to myocardial fibrosis and increased myocardial stiffness induced titin post-translational modifications. Titin is a big size sarcomeric protein involved in myocardial stiffness. Titin is produced in two main isoforms: the longer N2BA and the shorter N2B. In HFpEF patients, the N2BA/N2B ratio is altered. Oxidative stress and reduced nitric oxide (NO) production, are associated with impaired nitric oxide/cyclic guanosine monophosphate/protein kinase G (NO/cGMP/PKG) pathway and N2B titin post-translational modification ,62. MoreMicrotubules have a main role in the cardiomyocytes and myocardial compliance. In failing heart, microtubules are detyrosinated and mechanically uncoupled, determining increased viscoelastic resistance to diastolic stretch . The rolThere is a relationship between CMR parameters, such as native myocardial T1 and ECV, and invasive parameters . Omori eThe CMR importance in HFpEF also lies in its ability to predict patients’ prognosis and outcome. Poyhonen et al. found that HFpEF patients with CMR positive for LGE showed an increased risk of adverse events, including death . Kato etCMD is a condition characterized by coronary flow reserve (CFR) impairment in absence of coronary epicardial obstruction or unaltered flow fractional reserve (FFR) ,83,84,85As previously discussed, HFpEF related CMD is sustained by microcirculation histopathological alterations . MicrovaAnother important morphological alteration is microvascular hyperpermeability that has been observed in HFpEF related comorbidities, as well as in HFrEF and ischemia-reperfusion injury. Several molecular components are involved in the microvascular wall dysfunction. In this regard, the glycocalyx alteration promotes the adhesion molecules formation and neutrophils migration ,99. Pro-The lymphatic vascular system may be involved in the pathophysiology of HFpEF, contributing to the different myocardial histopathological findings observed in this condition . In partKato et al. study was the first to demonstrate the CFR impairment in HFpEF. They used phase contrast cine-magnetic resonance imaging for a non-invasively calculation of CFR, obtained from the ratio of coronary sinus blood flow with and without adenosine triphosphate (ATP) infusion . ResultsSystemic inflammation is an important pathophysiological mechanism involved in the HFpEF onset and progression. The HFpEF presence and associated conditions have been demonstrated in many systemic inflammation disorders, such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel diseases . MoreoveEpicardial adipose tissue is enhanced in hyperaldosteronism, hypercortisolism and hypothyroidism conditions. The inflammatory-metabolic HFpEF paradigm recognizes the epicardial adipose tissue as a target of systemic inflammation. Epicardial adipose tissue is subjected to several changes, through which it induces many alterations to contiguous tissues, through a paracrine and/or vasocrine effect. The vasocrine route refers to a newly proposed way of intercellular signaling, whereby adipocytes accumulate around arteries and larger arterioles of striated or cardiac muscle and secrete proinflammatory cytokines into the periadventitial space around these vessels. Under inflammation stimuli, cardiac mesenchymal cells are prone to differentiate in adipocytes, promoting the epicardial adipose tissue expansion. In this regard, the epicardial adipose tissue produces and releases several pro-inflammatory molecules through which it may cause CMD, myocardial fibrosis, myocardial electromechanical dysfunction and atherosclerosis progression, in epicardial vessels. The ventricular myocardium involvement is associated with hampered left ventricle filling and diastolic stiffness ,121, maiObesity prevalence in HFpEF patients is about 70%  and the Epicardial adipose tissue expression and inflammation are associated with circulating hormones levels. In this regard, natriuretic peptides reduce epicardial adipose tissue expansion and inflammation, as well as adipogenesis. Moreover, they counteract myocardial fibrosis and CMD ,132,133.From the hemodynamic point of view, inflammatory-metabolic HFpEF is characterized by sodium retention and glomerular resorption, systemic venous capacitance reduction and arterial blood pressure increase. The latter allows the distinction between inflammatory HFpEF from infiltrative and hypertrophic cardiomyopathies . Considering the relationship among other HFpEF risk factors and inflammation, RAAS activation induces monocytes migration and M2 macrophages myocardium infiltration, TGF-β production and myocardial fibrosis . MetabolVan den Hoogen et al.  found hiPrenner et al.  studied +/K+-ATPase) transporter activity [+/K+-ATPase activity, the regional analysis showed that this aspect was more evident at the apical level, and subsequently, at the anterior and anterior-lateral wall level. The inferior sectors showed no significant τi decrease. This model traces the metabolic changes in HFpEF and explains why there is much expectation in the use of drugs that act on an energy/metabolic level, such as sodium glucose co-transporter-2 (SGLT-2) inhibitors, in patients with HF and metabolic dysregulation [Regarding the metabolic aspect, CMR may provide further information in HFpEF. Through the hyperpolarized carbon-13 CMR spectroscopy, an increase in alanine and bicarbonate and no increase in lactate have been demonstrated . Alanineactivity ,145. Altgulation ,145,146.The pathophysiology complexity and the lack of specific therapy against HFpEF made this syndrome particularly prone to a personalized and precision medicine approach. The target is to define the HFpEF genetic and molecular basis, in order to predict the pathophysiological and clinical evolution, the patient’s outcome, as well as to find possible innovative therapeutic targets . Hahn etThe importance of epigenetic has been recognized recently and several studies focus on microRNA profiles that characterize HF subtype or stage. MicroRNA have a main role in the epigenetic modulation of gene expression acting at post-transcriptional moment. The presence of circulating microRNA demonstrate that they are released by cells after death probably acting as paracrine molecules. MicroRNA are involved in the different pathophysiological and histopathological aspects, which characterize HFpEF. Moreover, differences about microRNA targets, between HFrEF and HFpEF, have been observed regarding two pathways involving fatty acids biosynthesis and extracellular matrix receptors expression. The rationale of these observation is sustained by several histopathological findings that characterize HFpEF, as the minor expression of extracellular matrix destruction enzymes, such as metalloproteinase-2, and the enhanced expression of pro-fibrotic markers, such as galectin- 3 and the soluble type of IL-1 receptor like 1 (ST2) ,150. SevMacrophages are involved in tissue remodeling taking part in inflammatory response. Early phase of HFpEF is characterized by systemic inflammation outspread and the subsequent cardiac inflammation. In this context, macrophages, according with their polarization state, mediate the interaction between inflammatory cells and cardiac cells contributing to the cardiac remodeling ,154,155.Other microRNAs are involved in proteins post-translational modification such as the ubiquitin induced proteolysis regulation . miR-126Gender differences in HFpEF patients have been observed in term of microRNA expression. Florijn et al. demonstrated that plasma miR-224 and miR-452 are predominantly expressed by HFpEF diabetic women, instead of diabetic HFpEF man. Moreover, miR-34a is associated with kidney disease, diabetes mellitus and gender in patients with diastolic dysfunction, while decreased miR-34a, -224 and -452 plasma levels have been observed in diabetic patients with diastolic dysfunction and diabetic women with glomerular filtration rate under 60 mL/min . The defAngiotensin II receptor type 1 (AT1) stimulation induces myocardial hypertrophy and fibrosis participating to HF worsening . RAAS inA revolution of HFrEF treatment was carried out by the angiotensin receptor neprilysin inhibitor LCZ696, that combine the two acting molecules, valsartan and sacubitril. By inhibition of neprilysin, sacubitril increases atrial natriuretic peptide (ANP), BNP and C-type natriuretic peptide (CNP) plasma levels , throughHF is associated with oxidative stress, which affects myocardium and vasculature. Recent evidence suggests that oxidative stress may be the link between obesity, diabetes mellitus, and related complications. In obese patients, there is an increasing level of reactive oxygen and nitrogen species that directly correlate with central adiposity. The cGMP pathway plays a fundamental role in regulating normal cardiovascular function and its lack in HFpEF subjects leads to endothelial dysfunction. Endothelial dysfunction is related to cGMP deficiency that is caused by insufficient stimulation of soluble guanylate cyclase (sGC) and impaired NO bioavailability. This promotes CMD, myocardial and vascular stiffness .Correction of myocardial PKG activity and cGMP pathway has been proposed as a target for specific HFpEF treatment, but Sildenafil related phosphodiesterase type 5 (PDE5) inhibition did not show difference compared to placebo, in HFpEF patients .The sGC stimulator Vericiguat was studied in the SOluble guanylate Cyclase stimulatoR in heArT failurE Studies (SOCRATES) programme . In the Considering the hypothesis of decreased NO availability in HFpEF, nitrates have been proposed to restore NO balance, in order to improve endothelial-myocyte paracrine signaling. The Nitrate’s Effect on Activity Tolerance (NEAT-HFpEF) trial was conducted to investigate the role of isosorbide mononitrates in HFpEF management . IsosorbThe inorganic nitrate pathway represents a different way to restore NO signal . Unlike A new class of antioxidant peptides named “Szeto-Schiller peptides (SS peptides)” is under investigation in HF. SS peptides belong to a class of antioxidant that bind to cardiolipin, an important phospholipid of the inner mitochondrial membrane. These peptides protect cardiolipin from oxidation and reduce mitochondrial oxidative damage. One of the most prominent of these peptides is elamipretide . In patients with HFpEF, elamipretide reduced left-ventricular end-diastolic volumes, compared to placebo . Proinflammatory cytokines, such as IL-1 are upregulated in HFpEF. They have an important role in the myocardial function impairment. In HFpEF, IL-1 inhibits L-type calcium channels, downregulates phospholamban activity and causes post-transcriptional changes in SERCA2a . CalciumMyocardial inflammation and fibrosis are target of cell therapy. In animal models Pirfenidone, antifibrogenic drug which target TGF-β signaling, inhibits left ventricular fibrosis and diastolic impairment . Its rol+ cells may be suitable for cell therapy to improve diastolic function in HFpEF. A pilot study with HFpEF subjects showed that treatment with CD34+ cells determined diastolic function improvement and NT- proBNP levels decrease [+ cell therapy in patients with HFpEF is currently under definition. Dysfunctional calcium handling in HFpEF causes impaired myocardial relaxation. Late inward sodium current (INa) is increased in HF, causing cardiomyocytes calcium overload. Ranolazine inhibits persistent or late INa in heart muscle [CD34 is a receptor expressed by bone marrow multipotent progenitor cells, which are reduced in patients with both HFrEF and HFpEF. CD34decrease . The rolt muscle . In the t muscle . As repot muscle ,198. It t muscle . In thisIn HFpEF patients, high heart rate predicts poor outcome at sinus rhythm, instead there are no clear correlations between worse prognosis and atrial fibrillation. This finding was confirmed in The Irbesartan in Heart Failure With Preserved Systolic Function (I-PRESERVE) trial  and the Sodium-Glucose Cotransporter 2 Inhibitors (SGLT2i) showed a striking reduction of cardiovascular events, in patients with T2DM. SGLT2i reduces renal glucose reabsorption, rise urinary glucose excretion and increases diuresis ,206,207.consecutio in HFpEF onset and progression is not well established. A comprehensive overview of mechanisms involved in myocardial alterations observed in HFpEF are summarized in HFpEF is a multifaceted and complex syndrome associated with global high mortality and morbidity rates and its prevalence is constantly increasing. Although it is well known the association among HFpEF and traditional cardiovascular risk factors, myocardial alterations and pathophysiological basis are not well defined yet. In fact, the definition of HFpEF includes a wide spectrum of different myocardial structural alterations. Myocardial hypertrophy and fibrosis, CMD, oxidative stress and inflammation are only some of the main pathological detectable processes at myocardium levels and their


To evaluate the utility of the splenial angle (SA), an axial angular index of lateral ventriculomegaly measured on diffusion tensor MRI color fractional anisotropy maps, in differentiating NPH from Alzheimer’s disease (AD), Parkinson’s disease (PD), and healthy controls (HC), and post-shunt changes in NPH, compared to Evans’ index and callosal angle.Evans’ index, callosal angle, and SA were measured on brain MRI of 76 subjects comprising equal numbers of age- and sex-matched subjects from each cohort of NPH, AD, PD, and HC by two raters. Receiver operating characteristics (ROC) and multivariable analysis were used to assess the screening performance of each measure in differentiating and predicting NPH from non-NPH groups respectively. Temporal changes in the measures on 1-year follow-up MRI in 11 NPH patients  were also assessed.p < 0.0001). SA performed the best in predicting NPH, with an area under the ROC curve of > 0.98, and was the only measure left in the final model of the multivariable analysis. Significant (p < 0.01) change in SA was seen at follow-up MRI of NPH patients who were shunted compared to those who were not.Inter-rater and intra-rater reliability were excellent for all measurements (intraclass correlation coefficients > 0.9). Pairwise comparison showed that SA was statistically different between NPH and AD/PD/HC subjects  using tools readily accessible in clinical practice and shows excellent test-retest reliability.• Splenial angle outperforms Evans’ index and callosal angle in predicting NPH from healthy, Parkinson’s disease, and Alzheimer’s disease subjects on ROC analysis with an area under the curve of > 0.98 and is sensitive to morphological ventricular changes in NPH patients after ventricular shunting.The online version contains supplementary material available at 10.1007/s00330-021-07871-4. Idiopathic normal pressure hydrocephalus (NPH) is characterized by the classic triad of gait apraxia, dementia, and urinary incontinence but confounding factors commonly exist , 2. NeurThe callosal angle (CA) is more specific and useful in discriminating NPH from other neurodegenerative disorders with overlapping clinico-radiological presentations of ventriculomegaly from brain atrophy, cognitive impairment, and/or Parkinsonism gait disorder , 5. A naDiffusion tensor imaging (DTI) fractional anisotropy (FA) maps are increasingly incorporated into clinical practice , 11 due Approval from the institutional ethics committee for each NPH, AD, and PD study was obtained. All participants gave informed consent.Twenty patients were prospectively recruited from the NPH program at a tertiary referral center. All patients were diagnosed based on key clinical and neuroimaging features according to established criteria in published international NPH guidelines , 12, incAnonymized brain MRI of nineteen subjects each from the AD, PD, and HC databases age- and sex-matched to the nineteen NPH patients were sought for inclusion in this imaging analysis. The AD and PD patients were diagnosed based on established clinical criteria and recruited from the memory and movement disorders clinics respectively of a tertiary referral center –16. AD w3; repetition time ms/echo time ms, 7700/134; plane, axial), (B) three-dimensional (3D) T1-weighted high-resolution magnetization-prepared rapid gradient-echo (MPRAGE)  and (C) DTI . All axial acquisitions were parallel to the central inter-commissural line through the anterior and posterior commissures (AC-PC) identified by the MRI technologists on sagittal scout images as per institutional clinical routine for more than two decades of our MRI service. NPH patients with follow-up brain MRI at 1-year from baseline scan were performed using an identical protocol on the same 3-T scanner.For the NPH cohort, brain MRI was performed on a 3-T scanner including the following sequences: (A) fluid-attenuated inversion-recovery (FLAIR) , (B) 3D MPRAGE , and (C) DTI . All axial acquisitions were parallel to the AC-PC line as above.Brain MRI of the AD, PD, and HC subjects were all acquired on 3-T scanners using similar MR protocols with the following parameters: (A) FLAIR . Intra-rater and inter-rater reliability were assessed by intraclass correlation coefficients (ICC)  for all measurements. The averaged measurements from the two raters were used for each group comparison (since the ICC indicated excellent inter- and intra-rater reliability). Fisher’s exact test for categorical variables and ANOVA test for continuous variables were used to compare the demographic data and MRI brain measurements. In view of the multiple comparisons made, pairwise comparison of EI, CA, and SA adjusted by the Benjamini and Hochberg method was carried out to compare differences in these measurements between HC, PD, AD, and NPH groups by controlling the false discovery rate, the latter being the expected proportion of false discoveries amongst the rejected null hypotheses. Receiver operating characteristics (ROC) analysis was carried out to evaluate the sensitivity and specificity of the three indices as screening diagnostic tools for NPH from each non-NPH group individually. The area under the curve (AUC) highlighted the discriminative property of each index. A stepwise multivariable logistical regression analysis was performed to predict NPH from non-NPH groups with brain MRI measures as independent factors. Student’s t test was carried out to compare the changes from baseline in EI, CA, and SA between NPH patients who underwent ventriculoperitoneal shunting and those who did not. All statistical tests were evaluated as two-sided tests and statistical significance was defined at p < 0.05.Data analysis was performed using R 3.6.2  between NPH and non-NPH groups. The SA best segregated NPH from non-NPH groups compared to CA and EI  where SA, CA, and EI were measured were generally < 1 mmriations , 19–21. riations  for SA mFuture work needs to be done to assess the reproducibility of SA by different raters across sites, its inclusion in radiological scales summarizing imaging features into structured scores for diagnosis or prognosis , 29, cliThe SA measured on axial DTI color FA maps is a simple radiological index reflecting a horizontal angular perspective of lateral ventricular dilatation in NPH. It shows promise in differentiating NPH from the ex vacuo ventriculomegaly of aging and neurodegenerative disorders and sensitivity to morphological changes before or after shunt surgery in NPH. Further work is needed to evaluate feasibility in translating its efficiency from DTI FA maps to other routine axial CT or MRI sequences.ESM 1(DOCX 99 kb)


Functionalization of nanogold with this peptide has been achieved by coating nanoparticles with a peptide-albumin conjugate consisting of heterogeneous molecules with a variable number of linkers and peptides. To reduce nanodrug heterogeneity we have designed, produced and preclinically evaluated a homogeneous and well-defined reagent for nanogold functionalization, consisting of a head-to-tail cyclized CGisoDGRG peptide (iso1) coupled via its thiol group to maleimide-PEG11-lipoamide (LPA). The resulting iso1-PEG11-LPA compound can react with nanogold via lipoamide to form a stable bond. In vitro studies have shown that iso1, after coupling to nanogold, maintains its capability to bind purified αvβ3 and αvβ3-expressing cells. Nanogold functionalized with this peptide can also be loaded with bioactive tumor necrosis factor-α (TNF) to form a bi-functional nanodrug that can be stored for three days at 37°C or >1 year at low temperatures with no loss αvβ3-binding properties and TNF-cytolytic activity. Nanoparticles functionalized with both iso1 and TNF induced tumor eradication in WEHI-164 fibrosarcoma-bearing mice more efficiently than nanoparticles lacking the iso1 targeting moiety. These results suggest that iso1-PEG11-LPA is an efficient and well-defined reagent that can be used to produce robust and more homogeneous nano-vectors for the delivery of TNF and other cytokines to αvβ3 positive cells.Gold nanoparticles functionalized with  In partculature . Studiesiso1-HSA/Au/TNF and iso1-HSA/Au/IL12 as anti-cancer agents, these formulations have important drawbacks related to the use of iso1-HSA for nanogold functionalization with isoDGR. First, this peptide-protein conjugate, which consists of albumin molecules with a variable number of linkers (6-7 linkers) and peptides (4-5 isoDGR peptides) per molecule , instead of iso1-HSA. We show that gold nanoparticles functionalized with this compound maintain their αvβ3 recognition properties and are stable. Furthermore, we provide experimental evidence that these nanoparticles can be used as carriers for delivering TNF to tumors and to induce more efficient anti-tumor effects than nanoparticles lacking the isoDGR targeting moiety.To overcome these problems, we tested the feasibility of an alternative strategy for nanogold functionalization with isoDGR based on the use of a homogeneous and well-defined reagent, consisting of a peptide-polyethyleneglycol-lipoamide conjugate (520 nm ∼1 unit/ml) (Aurion); MAL-dPEG11-lipoamide  and MAL-dPEG3-lipoamide  (Quanta Biodesign). Murine TNF was prepared as described previously  (Baxter); human natural αvβ3 integrin ; anti-polyethyleneglycol (PEG) monoclonal antibody (mAb), clone 26A04 (Abcam); bovine serum albumin (BSA) fraction-V (Sigma); gold nanoparticles  and treated with HBTU/DIEA (1 eq.) to obtain the fully protected cyclo-CGisoDGRG peptide. DMF was removed under vacuum and the resulting crude material was directly treated with TFA-based cleavage mixture  to obtain the unprotected peptide, which was recovered by precipitation in cold diethyl ether. The peptide was purified by reverse-phase (RP)-HPLC, using a Shimpack GWS C18 column , lyophilized, and stored at −80°C. Aliquots of the product were dissolved in water and stored at −80°C until use. The identity and purity of the product, called iso1 (see+): 546.22 Da; found: 546.27 Da] and RP-HPLC analysis (purity > 95%).Linear peptide CGe method  on a 2-Ciso1 see, were coIso1-PEG11-LPA was prepared by coupling iso1 to Mal-PEG11-LPA using a 1:1.2 molar ratio, as follows: 4 mg of iso1 in 100 µl of 0.4 M sodium phosphate buffer, pH 7.8, were mixed with 50 µl of acetonitrile . The resulting solution was chilled on ice and mixed with 7.7 mg of MAL-PEG11-LPA in 100 µl of 50% acetonitrile and left to react under gentle shaking at room temperature. The coupling reaction was monitored by RP-HPLC and stopped when complete reagent conversion occurred. In parallel, a control conjugate was prepared using cysteine in place of iso1. Coupling reactions were monitored by RP-HPLC using a Shimpack GWS C18 column  connected to Shimadzu Prominence HPLC . The final products were purified by RP-HPLC using a Jupiter C18 column , and the same mobile phases indicated above . The products were lyophilized, resuspended in water, and stored at −20°C. Furthermore, iso1 and Cys were also coupled to MAL-dPEG3-LPA, i.e., a compound with a shorter PEG chain, using the same procedures. The identity of each conjugate  was checked by mass spectrometry analysis using an LTQ-XL Orbitrap mass spectrometer (Thermo Fischer) (Fischer) .iso1 we mixed aliquots of colloidal gold  with 4–32 µg of compound 1 in 5 mM sodium phosphate buffer, pH 7.4 (100 µl). The mixture  was incubated at room temperature for 2 h. To saturate gold nanoparticles, we then added 0.5% HSA  and left to incubate for 10 min at room temperature. The product was then centrifuged at 13,000x g for 15 min. The pellets were resuspended in 5 mM sodium phosphate buffer, pH 7.33, containing 0.05% HSA (buffer A). The centrifugation/washing steps were repeated twice. The final product  was resuspended with 1 ml of buffer A and stored at 4°C.To functionalize gold nanoparticles with 1 and TNF  were prepared as follows: a solution containing 0.540 mg of compound 1 and 2.88 mg of TNF in 18 ml of 5 mM sodium phosphate, pH 7.4, was slowly added (1 ml/min) to 180 ml of 25 nm-nanogold, with pH adjusted to ∼7.5 with sodium hydroxide, and left to incubate for 40 min at room temperature under shaking. The product was mixed with 18 ml of 0.5% HSA in water (slowly added) and left to incubate for an additional 15 min, to saturate gold NPs. The mixture was centrifuged  and resuspended in buffer A (three times). The pellet was then resuspended with 1.8 ml of buffer A, filtered , and stored at −80°C. Control nanoparticles bearing compound 2 and TNF  were prepared following the above procedure, except that in this case, 0.360 mg of compound 2 was used.Bifunctional gold NPs bearing compound buffer A or 5 mM sodium citrate buffer, pH 6.0, respectively, as blanks. The concentration of coated-NPs was calculated by interpolating the absorbance values at 530 nm on a calibration curve obtained using uncoated nanogold .Absorption spectra of coated- or uncoated-NPs were recorded using an UltroSpec 2100 spectrophotometer (Amersham Biosciences), a 1 cm path-length quartz cuvette, and Transmission electron microscopy (TEM) analysis was performed using a TALOS L120C microscope (ThermoScientific) as described previously . Morphom1 or 2 in cell culture medium . After washing, the cells were further incubated for 4 h at 37°C, 5% CO2, and then photographed using a bright field microscopy.Human MSR3 melanoma cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza) supplemented with heat-inactivated 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin/streptomycin. Cells were seeded into a 96-well plate  and incubated for 24 h in complete cell culture medium. After medium removal, the cells were incubated with nanogold functionalized with compound 1-Au/TNF were checked using two sandwich assays based on αvβ3-and anti-PEG antibody-coated plates followed by an anti-TNF polyclonal antibody. The αvβ3/anti-TNF polyclonal antibody sandwich assay was performed essentially as described (binding buffer), were added to microtiter plates coated with or without αvβ3 (0.5–1 µg/ml). The binding of nanoparticles was then detected using a rabbit anti-TNF polyclonal antiserum , followed by a polyclonal goat anti-rabbit HRP-conjugate. Bound peroxidase was detected by adding the o-phenylenediamine chromogenic substrate. The anti-PEG antibody/anti-TNF polyclonal antibody sandwich assay was carried out essentially as described above using a microtiter plate coated without or with an anti-PEG mAb (5 µg/ml) in the capture step, and the anti-TNF polyclonal antibody in the detection step.The bifunctional properties of escribed . Brieflyin vitro bioassay based on TNF-induced cytolysis of murine L-M fibroblasts, as described , weighing 18–20 g, were challenged with subcutaneous injection in the left flank with 1.5 × 106 WEHI-164 cells. Six days later, mice were injected (i.v.) with nanodrugs in 0.9% sodium chloride. Tumor growth was monitored by measuring the tumor size with a caliper. Tumor volumes were estimated by calculating r1 × r2 × r3 × 4/3π, where r1 and r2 are the longitudinal and lateral radii, and r3 is the thickness of the tumor protruding from the surface of normal skin. Animals were sacrificed before tumors reached a volume of 1 cm3. Tumor sizes are shown as mean ± SE.All procedures on mice were approved by the San Raffaele Institutional Animal Care and Use Committee, according to institutional guidelines, and in compliance with national  and international law and policies (new directive 2010/63/EU). Murine WEHI-164 fibrosarcoma cells  were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 μg/ml streptomycin, 100 U/ml penicillin, and 0.25 μg/ml amphotericin-B. WEHI-164 cells were tested for isoDGR peptide that can be directly attached to gold nanoparticles, we have coupled cyclo-CGisoDGRG (iso1) to MAL-PEG11-lipoamide and MAL-PEG3-lipoamide, two cross-linking reagents with different polyethylene glycol chains , Cys-PEG11-LAP (compound2), iso1-PEG3-LAP (compound3), and Cys-PEG3-LAP (compound4), were purified by RP-HPLC. The identity of each product was confirmed by mass spectrometry analysis  NaCl  did not cause nanogold aggregation by itself and could efficiently protect nanoparticles from salt-induced aggregation  were selected for further studies, whereas nanogold functionalized with compounds 3 and 4 were not further investigated. Nanoparticles prepared with compound 1 were further stabilized with human serum albumin and characterized by UV-Vis spectrophotometric analysis. The results showed that these nanoparticles were homogeneous, not aggregated, and resistant to salt-induced aggregation  could bind membrane-associated αvβ3, we tested the binding of this product to MSR3 cells, a human melanoma cell line that expresses high levels of αvβ3 and previously used for the characterization of iso1 (2 (2-Au) was also tested. Phase-contrast microscopy experiments showed granular dot-like staining in MRS cells incubated with 1-Au, but little, or not at all, with 2-Au , a larger batch of control nanoparticles lacking isoDGR using 16 µg/ml of TNF and 2 µg/ml of compound 2 (2-Au/TNF).To demonstrate that compound UV-Vis spectrophotometric analysis of both products showed single absorption peaks at 530 nm with a similar width, indicating that both products contained low (undetectable) amounts of aggregates . Transmi1 or 2 and TNF on the two nanodrugs, we measured the capability of each product to form molecular sandwiches with an anti-PEG monoclonal antibody (mAb) and an anti-TNF polyclonal antibody (pAb), or with αvβ3 and anti-TNF pAb. To this aim, we performed assays based on the use of microtiter plates coated with anti-PEG mAb or αvβ3 in the capture step and anti-TNF pAb in the detection step (seefor a schematic representation of the assays). 1-Au/TNF and 2-Au/TNF showed comparable binding properties when anti-PEG mAb-coated plates were used (left panel), suggesting that these nanodrugs were loaded with similar amounts of PEG-containing ligands and TNF. In contrast, only 1-Au/TNF was detected when αvβ3 was used in place of anti-PEG mAb in the capture step, as expected (right panel). Of note NPs functionalized with compound 3 (a ligand characterized by a shorter PEG length) and stabilized with TNF to prevent aggregation (3-Au/TNF) bound αvβ3 less efficiently than 1-Au/TNF  was then investigated. To this aim, 1-Au/TNF and 2-Au/TNF were left to incubate at 4°C or 37°C for various times (from 1 to 27 days) and then analyzed using the anti-PEG mAb/anti-TNF pAb and αvβ3/anti-TNF pAb sandwich assays. No significant changes occurred upon nanodrug storage for 9 days at 4°C or 3 days at 37°C (iso1 (the latter via lipoamide) can be stored at 4°C for at least 27 days without loss of function. Furthermore, no significant changes occurred upon nanodrug storage for > 1 year at –80°C  were cured with 1-Au/TNF, whereas only one out of six mice (17%) was cured with 2-Au/TNF  of TNF-bearing nanogold, at least as judged from the loss of animal body weight.Finally, we analyzed the anti-tumor activity and toxicity of 2-Au/TNF , suggest2-Au/TNF , suggestiso1-PEG11-LPA. This reagent consists of head-to-tail cyclized CGisoDGRG peptide (iso1) chemically coupled via its thiol group to maleimide-PEG11-LPA cross-linking reagent. Remarkably, the coupling reaction leads to the formation of a succinimide ring between peptide and PEG11-LPA cyclohexane-1-carboxylate (sulfo-SMCC) as cross-linking reagent, has a higher affinity and selectivity for αvβ3 than iso1 alone, thanks to the presence of the succinimide ring  that can react via lipoamide with nanogold to form a stable gold-thiol bond , maintained its capability to bind purified αvβ3 and to recognize MRS melanoma cells, an αvβ3-expressing cell line previously used for the characterization of isoDGR-containing compounds  bearing both targeting and effector moieties.This view is supported by the results of ompounds . Gold naDrug stability studies have shown that these bi-functional nanodrugs can be stored up to three days at 37°C, or >1 year at 80°C, in 5 mM sodium phosphate buffer, pH 7.33, containing 0.05% HSA, with no loss of activity in terms of αvβ3-binding and TNF-cytolytic activity. This result indicates that the new strategy used for nanogold functionalization herein described allows the production of stable nanodrugs.in vivo studies, performed using WEHI-164 fibrosarcoma-bearing mice, show that nanoparticles functionalized with iso1 and TNF according to this strategy (1-Au/TNF) could induce tumor eradication in WEHI-164 fibrosarcoma-bearing mice more efficiently than nanoparticles lacking the iso1 targeting moiety (2-Au/TNF). This result supports the hypothesis of an active targeting mechanism mediated by iso1 in the case of 1-Au/TNF. Given that WEHI-164 cells do not express αvβ3 receptors, these results also suggest that the improved anti-tumor activity of 1-Au/TNF was mediated by targeted delivery of TNF to a tumor stroma component, likely to αvβ3-positive endothelial cells. Notably, the iso1-mediated targeting mechanism increased the anti-tumor activity of TNF without increasing its toxicity, as judged from the even lower loss of body weight caused by 1-Au/TNF compared to 2-Au/TNF. This suggests that the targeted delivery of TNF-nanogold to tumors with iso1 can increase its therapeutic index.The results of iso1-PEG11-LPA is an efficient, well-defined, and robust reagent that can be used for nanogold functionalization with isoDGR and for enabling αvβ3 recognition by cytokine-bearing gold nanoparticles. This novel peptide-linker conjugate can overcome the need to use heterogeneous peptide-albumin conjugates, previously used for coupling the targeting peptide to nanogold (In conclusion, nanogold . The sam


What appears to be emerging from diverse pathologies in different organ systems is a common theme where pro-oxidative stressors generate oxidized glycerophosphocholines with PAF agonistic effects, which then trigger more enzymatic PAF synthesis via the PAF receptor. A downstream consequence of PAF receptor activation is the generation and release of MVP which provide a mechanism to transmit PAF as well as other bioactive agents. The knowledge gaps which when addressed could result in novel therapeutic strategies are also discussed. Taken together, an enhanced understanding of the PAF family of lipid mediators is essential in our improved comprehension of the relationship amongst the diverse cutaneous, cancerous, neurologic and systemic pathologic processes.Described almost 50 years ago, the glycerophosphocholine lipid mediator Platelet-activating factor (PAF) has been implicated in many pathologic processes. Indeed, elevated levels of PAF can be measured in response to almost every type of pathology involving inflammation and cell damage/death. In this review, we provide evidence for PAF involvement in pathologic processes, with focus on cancer, the nervous system, and in photobiology. Importantly, recent insights into how PAF can generate and travel  The lyso GPC species then is acetylated using acetylCoA by an acetyltransferase  with PAFR activity is dependent upon amounts of GPC substrate, pro-oxidants and antioxidant defenses. It should be noted that the majority of ox-GPC species have not been structurally characterized to allow quantitation using mass spectrometric techniques. Hence, we believe that the most accurate manner to measure PAF is via its biochemical effects such as intracellular calcium mobilization responses or release of cytokines such as IL-8 in genetically engineered cell lines with/without the PAFR   the GPC –15. Oxidthe PAFR , 16–18.Once PAF is generated, it can either reside in the cellular membranes and potentially act upon the cell itself or to neighbor cells in juxtacrine fashion , 20. SomBecause elevated PAF levels can be measured in many diseases, and exogenous PAF can mimic many aspects of disorders, PAF has been implicated in many processes. However, no actual diseases have been demonstrated to be due entirely to the presence or lack of the PAF system. The picture that is emerging is that the PAF system appears to serve as a modulator of pathologic processes. There are three areas that we would like to focus this review upon- the role of PAF in cancers and cancer therapy responses, central nervous system pathologies, and the effects of ultraviolet B radiation. Given that the skin is a complex organ that has epithelial, mesenchymal, immunologic, and neuronal components, all of these areas link to the overall theme of the epilipidome. Moreover, the three areas are connected to what we believe is a common three-part process. First, ROS from various agents generate small levels of PAFR agonists. Second, these PAFR agonists act upon the PAFR resulting in cellular activation and generation of additional PAF enzymatically, potentially allowing a PAFR amplification response. Finally, aSMase activation results in the formation and release of MVP carrying PAFR agonists to other sites. Activation of PAFR in target organs then mediate further pathology. This process provides several therapeutic targets to include antioxidants, PAFR antagonists, aSMase inhibitors as well as agents that block down-stream effects such as cyclooxygenase-2 (COX-2) inhibitors.The ability of the PAF-PAFR signaling to induce a robust systemic pro-inflammatory, pro-proliferative, and delayed immune suppressive responses, implicated in various pathological conditions, rationalized its exploration in cancer development as many malignant cells were identified to express PAFR –5, 30. NGiven that ectopic PAF-AH expression in KS-Imm human Kaposi’s sarcoma cells, or B16F10 melanoma cells resulted in reduced neoangiogenesis and tumor growth in their respective SCID and C56Bl/6J hosts, and that IFNγ-stimulated PAF synthesis enhanced the invasiveness of F10-M3, a clone of B16F10 melanoma line , 36, thein vitro models, as well as in clinical settings of cancer patients. Data from in vitro cellular models indicate that regardless of the anatomical origins, genetic backgrounds or the mechanisms involved, activation of tumoral-PAFR or PAFR overexpression accelerate the proliferation, aggressiveness, migration and invasion compared to respective control cells in various cancer models  tumors and treated with or without PAF antagonist and added over glass coverslips. Increased spreading of macrophages was observed within a shorter period after tumor cell implantation. However, as tumors continued to grow, the spreading of macrophages derived from the normal (vehicle-treated) mice decreased, but macrophages from PAF antagonist-treated mice maintained the elevated levels of spreading  tumor lines , cervical cancer , and squamous carcinoma (SSC78 and SSC90) cell lines compared to their respective unirradiated controls  inhibitors or depleting antibodies against Tregs , 42. Concontrols . Treatmecontrols .via these therapeutic agents impede treatment efficacies in a PAFR dependent manner, and that PAFR serves to mediate pro-survival responses to these agents.Importantly, our studies have also detected an increased level of PAFR activity in the perfusates collected post-chemotherapy compared to pre-chemotherapy in melanoma patients using the isolated limb chemoperfusion . Higher Several groups including ours have proposed PAFR as a promising target to not only inhibit tumor growth but also to increase the efficacy of therapeutic agents. This hypothesis has been tested in multiple experimental models demonstrating that genetic blockade of the PAFR (via studies in PAFR KO mice or PAFR shRNAs) or pharmacologic PAFR antagonists significantly reduced tumor burden, increased murine host survival, as well as augmented the effectiveness of therapeutic agents compared to their respective control groups , 64, 65.At present, it is unclear whether the PAF system plays an important functional role in the nervous system. Though PAF injection into the skin has been reported to be painful –71, muchAlthough the role of PAF in CNS function is unknown, the PAF system has been implicated in multiple CNS pathological states. Elevated PAF levels have been detected in and appear to correlate with severity of several CNS diseases. The association of elevated PAF with Alzheimer’s Disease (AD), multiple sclerosis (MS), cerebral infarction, cerebral ischemia-reperfusion injury, and spinal cord injury has been recently reviewed . ModeratIn addition to increases in PAF levels, a reduction in PAF-AH activity was observed in PD  and MS pIn vivo administration of a PAFR antagonist (LAU-0901) in a mouse model of temporal lobe epilepsy attenuated seizure susceptibility and neuronal hyper-excitability as well as reduced hippocampal damage  echanism –107. Ratechanism . There i calcium , 107. Ho calcium , 107.At present, there are no reports as to the identification of PAF-containing MVP in the CNS. However, microglia release MVP containing IL-1β within the CNS upon ATP stimulation . SimilarIn summary, multiple studies have implicated the PAF system in a number of neuroinflammatory and neurodegenerative conditions. As these mechanisms are further elucidated, there is potential for PAF and PAFR targeted therapeutics for various CNS disorders. In particular, one area which has significant promise as a therapeutic target relates to MVPs, which are likely elevated in a broad range of CNS pathologies. It is presumed that an enhanced understanding of the PAF system in CNS disorders would be reflected upon the peripheral nervous system.There is accumulating data suggestive that the PAF system could play a significant role in cutaneous pathophysiology, in particular how the skin responds to exogenous environmental stressors . Cell tyUltraviolet B radiation  is a pro-oxidative stressor which exerts profound cutaneous effects. Though only appreciably absorbed in the epidermis, sunlight’s “burning rays” are critical for vitamin D metabolism, yet, also generate sunburns as well as both melanoma and non-melanoma skin cancer –126. Inavia ox-GPCs , Veteran’s Administration Merit Award 5I01BX000853 (JT).The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The views expressed in this presentation are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.


Tumor necrosis factor-alpha (TNF-α) inhibitors are frequently used for the management of type 1 helper T-cell (Th1) immune-mediated chronic inflammatory conditions such as psoriasis and Crohn’s disease. Although TNF-α inhibitors are usually well-tolerated, various cutaneous side effects are frequently observed, including eczematous or atopic dermatitis-like eruptions. It is postulated that the attenuation of the Th1 immune pathway with TNF-α inhibition causes a shift towards a type 2 helper T-cell (Th2) immune response, leading to the development of skin lesions grossly and histologically consistent with the Th2 mediated disease atopic dermatitis. Herein, we describe the development of an eczematous eruption in two patients with a history of Th1-mediated disease after months of therapy with a TNF-α inhibitor. An eczematous or atopic dermatitis-like eruption is a common adverse cutaneous event in patients receiving tumor necrosis factor-alpha (TNF-α) inhibitor therapy . TreatmeCase 1A 64-year-old woman was seen for treatment of chronic plaque and inverse psoriasis with 15% body surface area involvement. The patient had a five-decade history of psoriasis and had failed multiple therapies, including methotrexate, clobetasol, fluocinonide, tacrolimus, narrow band UVB phototherapy, and tar. Her pertinent past medical history included seasonal allergies and adult-onset asthma. She had no personal history of atopic dermatitis nor family history of atopy.The patient was started on 40 mg adalimumab biweekly after an 80 mg initial dose, which resulted in complete control of her psoriasis. After four months of therapy, she developed a pruritic vesicular rash on her hands. She denied changes to personal care products used. Cutaneous examination revealed deep-seated vesicles within pink plaques with a fine-scale involving the palmar hands bilaterally Figure .A punch biopsy of the left hypothenar skin showed a mildly acanthotic epidermis with spongiosis and focal lymphocytic exocytosis. Within the superficial dermis, a sparse perivascular lymphocytic infiltrate was noted without eosinophils Figure . PeriodiClinical and histologic findings supported the diagnosis of dyshidrotic hand dermatitis. Adalimumab therapy was not discontinued, and the patient was started on halobetasol 0.05% ointment twice daily. The hand rash resolved after two weeks of topical corticosteroids but flared after she discontinued medication.Case 2A 14-year-old girl presented for evaluation of a rash on the neck and arms. The patient was diagnosed with Crohn’s disease seven months prior and started treatment with adalimumab and methotrexate. After two months of therapy, she developed a pruritic rash on the dorsal neck and bilateral upper extremities that was not relieved by over-the-counter lotions and baby oil. Self-discontinuation of adalimumab and methotrexate for four weeks resulted in no significant improvement of her symptoms. Therefore, she resumed therapy with both medications before visiting our dermatology clinic. The patient has no personal history of atopy, but family history is notable for atopic dermatitis in the mother and a sibling.Skin examination demonstrated ill-defined erythematous scaly thin plaques and hypopigmented patches on the bilateral ventral forearms, antecubital fossae, upper arms, and nape of the neck Figure . A cliniAtopic dermatitis and psoriasis are chronic inflammatory skin conditions driven by opposing helper T-cell populations . WhereasThe recognition of TNF-α as a key promoter of systemic inflammation has led to the use of TNF-α inhibitors, such as infliximab, etanercept, and adalimumab in the treatment of psoriasis and Crohn’s disease ,8. HowevLike the patients in this report, the development of an eczematous or atopic dermatitis-like eruption following TNF-α inhibitor therapy is well-documented in multiple case reports ,3, prospInterestingly, a contrasting phenomenon has been reported where patients with atopic dermatitis developed psoriasiform eruptions after treatment with dupilumab, a monoclonal antibody against Th2 cytokines IL-4 and IL-13 -17. In tIn summary, physicians should be aware that TNF-α inhibitors may cause an eczematous eruption as a result of a Th1 to Th2 phenotype switch. In particular, patients with a strong personal or family history of atopy should be advised of this potential side effect. Typically, the eczematous eruption can be managed with topical corticosteroids without discontinuing TNF-α inhibitor therapy.


PED) and genomic inbreeding estimates was found for FROH (runs of homozygosity-based F coefficient) and FUNI (F coefficient based on the correlation between uniting gametes). FROH correlation with FPED tended to increase as the number of generations registered as pedigree increased. While lineage and gene contributions (Q) from Admixture software correlated, they showed poor direct compliance; hence, Q-value cannot be recommended as the estimate of pedigree-based lineage. All these findings suggest that the methods of genomics should be considered as an alternative or support in the analysis of population structure in conservative breeding that can help control inbreeding in rare horse populations.Traditionally, pedigree-based relationship coefficients were used to manage inbreeding and control inbreeding depression that occurs within populations. The extensive incorporation of genomic data in livestock breeding creates the opportunity to develop and implement methods to manage populations at the genomic level. Consequently, the realized proportion of the genome that two individuals share can be more accurately estimated instead of using pedigree information to estimate the expected proportion of shared alleles. To make use of this improvement, in this study we evaluated the genomic inbreeding measures in the Polish conserved cold-blooded horse population and compared the data with the traditional measures of inbreeding. Additionally, an ancestry fractions/proportions from Admixture software were tested as an estimate of lineage (ancestry coefficient) used for horses qualifying for the conservation program. The highest correlation of pedigree-based (F The accuracy of inbreeding estimations based on pedigree information depends on the precision of breeding documentation. However, mistakes often appear due to imprecise records and limited knowledge of the origin. Ancestry information is difficult to obtain even for some registered animals because 1) one or more paths of their pedigrees may trace to or through foreign herd books, and (2) early ancestry information may not be recorded electronically [ one or mIn recent years, the results of genomic and pedigree-based studies have been compared to estimate inbreeding, especially for populations in danger of extinction. Kardos et al.  used comCold-blooded horses appeared in Poland in the second half of the 19th century. It was related to the economic exigence and associated with the import of sires from Western Europe. The main breeds included Ardennes, Belgian and Breton, while the less used breeds included North Swedish, Russian, Døle and Mur-insulan, as well as single documented cases of Fjord and Canadian horses and Boulonnais and Jutland stallions ,7. In thThe pedigree information of these populations can be incomplete as the divisions into local breeds were removed in the second volume of the stud book . All indIn the genetic resources conservation program, only horses that have been entered in the stud book of the Polish draft horse, have an appropriate pedigree and have a conformation that conforms to the breed standard can participate. In the gene pool of Sztumski and Sokólski horses the most important contribution has come from Ardennes, Polish cold-blooded (z) and unknown (NN) horses, followed by Belgian horses. This influenced a relatively high number of founders, namely 1139 for Sztumskis and 1118 for the Sokólski population, with the effective number of founders of 156.9 and 111.4, respectively. Additionally, the data indicates that only one third  of the gene pool in both populations comes from local horses, and as much as two thirds from imports . It is kThe generation intervals for both populations were similar—7.46 years for Sztumski and 7.24 for Sokólski horses. The pedigree completeness for five generations was respectively 98.79 and 98.24 and for all known generations, namely 16, was 51.11% and 45% . From thThe aim of this study is to assess the concordance and mutual relationships between pedigree-based and single nucleotide polymorphism (SNP)-based inbreeding coefficients as well as between ancestry coefficients calculated using pedigree and genomic data. The results of this analysis will give a new toolset for the horse resources conservation program in Poland and will allow the assessment of the reliability and usefulness of genomic data for the maintenance of conserved horse populations.PED) and so called genomic inbreeding  measures expressed by four different coefficients. In this study we compared and analyzed in detail the inbreeding estimates calculated based on pedigree data  and Sztumski (n = 69), which were previously analyzed for genetic differentiation [The study of genomic inbreeding was based on 175 cold-blooded horses, belonging to two types, namely Sokólski  <1.0 × 10−6 in each breed separately were excluded. The final SNP panel of 52,023 markers was scattered across the horse genome  with an average inter-marker distance of 43.0 kb. For ROH detection, the filtered SNPs set was remapped to the EquCab3.0 (https://www.ncbi.nlm.nih.gov/assembly/GCF_002863925.1/) genome assembly using the UCSC Genome Browser and Lift Genome Annotations tool. This removed another 109 SNPs that did not map to the newer assembly. Based on these filtered data, four inbreeding measures were calculated, similarly as in our previous study [GRM), (ii) method-of-moments F coefficient estimate (similar to FIS), (iii) F coefficient based on the correlation between uniting gametes (FUNI) [ROH, including ROH segments with lengths above 1 Mb) [Genomic inbreeding was evaluated based on Neogen Equine Community array  data, including genotypes for 65,157 SNPs with an average inter-marker distance in EquCab2.0 genome of 36.3 kb. SNP data were filtered as previously described . In brieus study ,17: (i) s (FUNI) , and (ivve 1 Mb)  using Plve 1 Mb) . The firve 1 Mb)  and evalve 1 Mb) . Comparison among various measures of inbreeding was made using Spearman’s rank correlation (rho) coefficients following data distribution evaluation with the Shapiro–Wilk test. Statistical significance of differences in F coefficient between two analyzed horse types was assessed using an ANOVA test. Statistical testing was done using JASP software .Moreover, a Q-value (ancestry fractions/proportions) from Admixture software , calculaPED), is presented in The distribution of the inbreeding coefficient in the reference population of the Sztumski and Sokólski cold-blooded horses, estimated on the basis of the pedigree information  is very similar to the distribution in the entire population of Sztumski and Sokólski cold-blooded horses . NeverthThe pedigree completeness index per each generation show that in the first two generations, 100% of the ancestors are present. In the third and next generation there is an increasing loss of ancestors, most often due to the fact that they are individuals of foreign breeds whose pedigrees, with the assumptions, are not entered into the database.The number of available ancestors’ data in our pedigree information is presented in The visible decrease in the number of pedigree information from the sixth generation results from the appearance of numerous imported ancestors–founders, i.e., whose data was not entered into the database . It is aPED data showed (expected in breeding populations) an increasing trend, while genomic measures rather showed a declining tendency  registered in the pedigree data . While atendency .PED, genomic measures of inbreeding either under (FGRM) or overestimated the population inbreeding  and FUNI (2–5.1%) coefficients. Negative inbreeding values (outbred) were observed exclusively for FGRM, suggesting the presence of some statistical artifacts.When compared to Fbreeding . FPED vaComparison of all F coefficients among the analyzed horse types (SOK and SZTUM) did not show any statistically significant differences . SimilarROH and FUNI  and the weakest between FPED and FGRM . FPED showed the strongest correlation with FROH and FUNI with Spearman’s rank of 0.443 and 0.430 (p < 0.001), respectively  (While taking into account the number of generations registered in the horse pedigrees (pedigree depth), correlations of Ferations . This co= 0.028) .p < 0.001)   and show< 0.001) .PED) using methodology proposed by Wright [In this study we analyzed and performed various comparisons between different measures of inbreeding, including pedigree-based coefficient and genomic measures of inbreeding. It is well known that the mating of related individuals results in the inbreeding of an offspring . In closy Wright  or more y Wright . When pey Wright . Inbreedy Wright , by methy Wright , from thy Wright , from siy Wright , based oy Wright  or from y Wright ,28. A hiy Wright . To avoiROH showed the strongest correlation with FPED data. In this analysis we used F calculated based on ROH with a length >1 Mb. This was because we wanted to capture a total animal autozygosity, including relatively distant (ancient) incidents of inbreeding which often cannot be shown in the pedigree analysis due to the lack of information. ROH are defined as contiguous homozygous regions in the genome where the two haplotypes inherited from the ancestors are identical by descent [PED and FROH tended to increase along with the increase of the number of generations registered in pedigree data. Similar observations were also made by other authors [ROH values presented in this study are visibly lower than ones presented in our previous work [UNI and FPED. FUNI is defined as a correlation between genetic effects that gives more weight to homozygosity at rare alleles [UNI is directly related to the definition of Wright [UNI can be an appropriate inbreeding measure to estimate inbreeding depression, whereas in scenarios of small population sizes, FROH may be more appropriate [UNI appears to be simpler to calculate than FROH, it can be also considered as a useful measure of inbreeding in conserved horse populations. Unexpectedly low correlations were found between FGRM and FROH, especially when FGRM belongs to the same group of methods as FUNI, and is based on covariances between genetic effects [Recently, an inbreeding coefficient derived from ROH was developed and shown to be optimal for the estimation of genome-wide autozygosity and for detecting inbreeding effects ,28,32,33 descent . Previou descent . This wa authors . The FROous work . This waous work . However alleles . FUNI isf Wright . It was ropriate . While FROH is the most similar to the pedigree-base estimates measure of inbreeding and we demonstrated its ability to reflect an ancient inbreeding. We also presented FUNI as a congruent measure of inbreeding, with a similar range and values as FPED and FROH. While lineage and gene contributions from Admixture software correlated, it showed poor direct compliance, hence Q-value cannot be recommended as the estimate of pedigree-based lineage. All these findings suggest that the methods of genomics should be considered as an alternative or support in analysis of population structure in conservative breeding and can help in the control of inbreeding in rare horse populations.In this study, based on a conserved horse population, we showed the usefulness of genomic data to assess population inbreeding with relatively high conformance to pedigree-based estimates. We confirmed previous observations that F


A researcher should only be an author on a paper if they have contributed to it in a substantive way. I recently read Nancy Kleckner’s appreciation of her late husband, Guido Guidotti, who died in August 2021 after a long and distinguished career at Harvard University . I stronGuidotti also had interesting ideas on the authorship of scientific papers. Back in 1960, when he published his first paper, only those researchers who had had a significant role in generating the data were listed as an author, and it was not uncommon for the papers from PhD theses to have just one author because a PhD thesis was meant to be an independent piece of work. The community of scientists in any field of research was so small back then that people in the field would likely know that a first-time author was a PhD student in an established scientist’s laboratory.In accord with this policy, my own PhD papers were single-authored, as were those of several of my lab colleagues. At first our thesis supervisor – a superb electrophysiologist called Allen Selverston – only signed papers from his lab when he had actually participated in collecting the data. However, shortly after I completed and published my thesis papers in the mid-1970s, it became almost unheard of to have single-authored papers from students and postdocs. So, in electrophysiology, as in other areas of biology, it became customary for lab heads to be the last author on papers from their lab. This progression has also been described explicitly for the field of meiosis .However, Guidotti maintained his policy of only putting his name on a paper if he had done some amount of bench work to collect the data as he felt that this was the right thing to do, and he adhered to this policy long after it was no longer in his interest to do so. Indeed, according to a profile of Guidotti that was published in the newsletter of the American Society for Cell Biology (ASCB) in 2003, he only abandoned this policy when it started to cause problems with funding agencies: "Guidotti says his days as an unacknowledged collaborator ended abruptly in 1985 when he was called on the carpet before an NIH study section. 'They said I was plagiarizing by using these articles that were published without my name on them,' Guidotti recalls. 'So now I put my name on all the papers, even the ones I didn’t work on.'" (The ASCB profile of Guidotti is included as an appendix in Similarly, I have refrained from signing papers on which I had literally no input. For example, when one of my postdocs wrote a paper on capacitance in cables , I decliOf course, the world has gone awry in numerous ways, many of them far more important than the question of who signs the papers from our labs. Meanwhile, some of the old ways are best: we should only be authors on papers when we have contributed in a substantive way. That should go without saying, but as with so much in today’s world, ethical principles seem increasingly at odds with practice.Living Science collection.This essay is part of the


The majority of papers  were cited between 1 and 10 times, whilst less than 7% of papers received more than 40 citations. Discussion: It is desirable that papers on pediatric dentistry increase in the pediatric scenario, allowing the two related disciplines to intertwine more in the future.Background: Pediatric dentistry shares many skills with pediatrics. This review evaluates the amount of literature on pediatric dentistry in the first 30 pediatric journals classified by the Web of Science in 2019. The aim was to perform a quantitative analysis of the main dental topics addressed. Methods: A scoping review with the PRISMA-ScR criteria was performed. The Clarivate Analytics Journal Citation Report was consulted for journals ranked in the category “Pediatrics” in 2019. Papers were searched in PubMed using an ad hoc prepared string. Results: A total of 504 papers were included. Papers on dental hard tissues were the most prevalent (45.6%), followed by dental public health (23.2%), orofacial development (15.3%), soft tissues related conditions (12.3%), and orofacial trauma (3.6%). Increasing trends have been observed for total papers published (R Oral health is an essential part of overall health and wellbeing. Good oral health needs not only sound teeth and periodontium, but also the wellbeing of all structures of the mouth, jaw, oral, and peri-oral tissues. Most oral health conditions are largely preventable in children and can be treated in their early stages. Caries represent one of the most common non-communicable diseases  and the The prevalence of untreated dental caries is high worldwide. Data from the Global Burden of Disease (GBD) 2015 show that 3.5 billion people live with dental health-related issues globally . NeverthDentists are the specialists of oral health. In the majority of countries, they are enrolled in a separate faculty from medical students. During the past decades, dentistry developed in many hyper specialties, and pediatric dentistry is one of them. Anyway, this evolution leads to an estrangement of highly related branches of medicine like pediatric dentistry and pediatrics ,29. ThesThe scoping review evaluates the amount of literature published on pediatric dentistry in the first 30 pediatric journals classified by the Web of Science (WOS) in 2019. In addition, a quantitative analysis of the main dental topics addressed over time will be presented.A scoping review was designed and performed to investigate the literature addressing dental topics published in pediatric journals. The review followed the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines  32]..32].Type of study: Review/meta-analysis, guidelines/consensus paper, interventional studies, observational studies, case report/case series;Publication languages: No languages restriction applied;Time of publication: No time restriction applied;Topics: Any papers regarding pediatric dentistry issues as the primary or not primary outcome, including diseases of hard and soft oral tissues, orofacial trauma, orofacial development, dental public health;Outcomes considered: Number of papers, types of studies, topic addressed, number of citations.The inclusion criteria were:The Clarivate Analytics Journal Citation Report (JCR) was consulted in April 2021 (S.C.) to retrieve journals ranked in the category “Pediatrics” in 2019, which was the last year JCR had been released at that time. The top 30 journals with the highest impact factor score and indexed in Medline were selected. Papers were searched in Medline electronic database via PubMed from the inception until 31 May 2021, using the following search string:journal title”[Journal]). In case a journal has changed its name over time, the search string with the previous journal titles has been also searched  AND . Four authors  independently examined all the abstracts to establish whether each paper should or should not be included in the scoping review. Disagreements were resolved through discussion in doubtful cases. Where resolution was not possible, another author was consulted (M.G.C.).The output of the references searched was uploaded into ExcelA structured template for charting the data was designed (G.M.T.). The data extraction form was realized without masking the name of the journal, title, and authors.The data extraction form contains and summarizes the main characteristics of each paper: journal title, journal impact factor, total journal citations, paper title, authors, publication year, PubMed Uniform Resource Locator, country, area, topic, type of study, oral health primary outcome, number of citations.Hard tissues: caries prevention, caries treatment, caries epidemiology, developmental defects of enamel (DDE) and dentine;Soft tissues: oral aphthae, oral infections, gingivitis, and other soft tissues disease;Orofacial trauma: dental trauma, jaws trauma, combined trauma , suspected abuse;Orofacial development: orofacial anomalies in syndromic and not-syndromic patients;Dental Public Health: health insurance, health services, pediatrician’s role.Dental topics were grouped into five areas as follows:Moreover, papers were divided into two categories, those with oral health as the primary outcome, and those including an oral health issue within other outcomes.Finally, an author (C.S.) checked every extraction for accuracy and completeness.®, Redmond, WA, USA). Included studies were not subjected to critical appraisal, since the objective of the review was to evaluate the amount of literature published on pediatric dentistry in pediatric journals.Data extracted from the studies were exported to an Excel spreadsheet  were published in “Pediatrics”, which was also the journal that published over time the highest numbers of papers , 17% (n = 87) in “The Journal of Pediatrics”, and 31% (n = 156) in other three journals . Among the other journals, from twenty of them, less than 25 papers each were retrieved, and from the last five, no paper was found. Journals addressing general pediatric medicine had higher numbers of papers on dental topics than those publishing papers related to a specific field within pediatrics  with no significant differences (χ2(6) = 0.051 p > 0.05), although the rise in numbers of papers on dental issues has been delayed, starting from the 1990s. Within papers on dental issues, those featuring hard tissues and public health issues mostly contributed to the publication trend in the last three decades  for the two dentitions was investigated. Caries data were obtained from the Global Burden Diseases Database available from 1991 . Caries n = 292, 57.9%) were cited between 1 and 10 times, whilst less than 7% of papers received more than 40 citations. A small group  of papers have no citations, and only four papers have obtained more than 100 citations , with caries prevention and health insurance as the most cited topics . Papers focusing on oral health as the primary outcome were cited more than those in which it was not, except for soft tissues and orofacial development areas. The highest average number of citations per paper was observed for public health area (mean = 17.3), followed by hard tissues (mean = 12.0), soft tissues (mean = 11.9), orofacial development (mean= 8.8), and orofacial trauma (mean = 4.6) area. All areas, except for orofacial trauma, included papers with a number of citations greater than 40; nevertheless, orofacial trauma did not include any paper with no citation.The majority of papers (itations . The citThis scoping review highlights the shortage of papers on pediatric dentistry issues in pediatric journals. In fact, although papers on dental topics increased over time, especially in the last two decades, the number of papers remains small if compared to the impressive increase of the overall number of papers in the pediatric journals considered.A considerable asymmetry in the distribution of papers on dental issues among the thirty journals considered was observed. The majority of papers had been published by only five general pediatric journals, while those dedicated to specific areas seem less interested in publishing oral health issues. Surprisingly, only one paper was found in journals dedicated to childhood obesity, even if multiple aspects and shared risk factors link oral pathologies and the systemic condition ,35. ThisObservational studies were the most prevalent type of study in all dental areas considered. Observational studies offer advantages as the relatively quick and low-cost carrying-out, and can be used to study multiple outcomes simultaneously. However, the main limit is that they cannot differentiate between cause and effect or within the sequence of events, and so they are not considered high-quality design studies. Nevertheless, because this study aims to measure risk factors or to collect exposure data related to a disease, as for many papers on dental topics, observational studies are often suitable .The most common topic of papers published in pediatric journals was caries prevention; on that note, the number of papers seems to follow caries prevalence variation over time. Although a high number of papers published on caries prevention in the last decade were found, caries data still highlights a high prevalence of lesions in primary teeth. This situation requires a great collective political, economic and medical effort, and the knowledge deriving from scientific literature can only represent the starting point. Moreover, an effort should also be advocated in improving communication and cooperation among practitioners who care for children to share multi-specialist knowledge .The majority of the papers on dental topics amounted to less than ten citations each. This data is in line with the number of citations found for the top 100 most-cited papers published in pediatric dentistry journals in 2019, where only seven papers were cited more than 100 times with a range between 42 and 182 citations . An inteSome limitations of this review need to be considered—firstly, the choice of the journals included. Papers published in the first top 30 out of 128 total pediatrics journals ranked in 2019 JCR were included. This choice could have excluded eligible papers and might have influenced the final results. Journals searched represent nearly 25% of all pediatric journals ranked in 2019 and were all indexed in the 2018 and 2020 JCR. Secondly, the use of a bibliometric ranking to select the journals avoids the introduction of selection bias. Within this limit, it can be assumed that the journals and papers selected can be considered representative for the category, and results provide a reliable, though restricted, overview of dental publications in pediatric journals that fits the purpose of this scoping review. Thirdly, this review also included papers in which the dental topic was not the primary outcome, but only one of the aspects considered. This choice led to an enlargement of the included sample by about a quarter. On the one hand, if this inclusion may seem a selection bias, on the other, it allows the inclusion of multidisciplinary papers that are extremely useful for a correct integrated management of young patients. A strength of this review was that it was the first, at best of authors’ knowledge, aiming to verify how much two related disciplines share scientific knowledge. This aspect is becoming more important day after day, due to knowledge advancement’s speed in medical fields, requiring constant updates not only on specific issues of each medical branch, but also on related issues. The scientific literature offers a unique chance to obtain constantly update information in an easy and economical way.In conclusion, this scoping review provides an overview of dental publications in the first 30 pediatric journals according to the WOS impact factor. The results show a small number of dental publications compared to the total amount of published papers. The areas of greatest pediatric interest in the dental field seem to be caries and dental public health-related issues. The papers in these two areas were even the most cited. It is desirable that papers on pediatric dentistry issues arise in number and interest in the pediatric scientific scenario, allowing the two related disciplines to intertwine more in the future.