Incubation of protonated NADH or NADPH with ortho-aminobenzaldehyde generates a novel fluorescent nicotinamide dihydroquinazoline condensate.

Incubation of reduced nicotinamide adenine dinucleotide (NADH) but not oxidized NAD+ with ortho-aminobenzaldehyde (oABA) generated an uncharacterized chromophore with an absorption peak characteristic of a dihydroquinazoline condensate. This chromophore is responsible for a non-specific signal in a diamine oxidase (DAO) activity assay based on the generation of fluorescent dihydroquinazoline structures directly from DAO substrates. Herein we show that at pH values below 3.0 the glycosidic bond of NADH/NADPH is broken releasing double protonated dihydro-nicotinamide (dihydro-NAM), which consequently condensates with oABA to a novel dihydroquinazoline chromophore and fluorophore, namely the 6- or 8-carbamoyl-5H,7H,8H,9H-10λ5-pyrido[2,1-b]quinazolin-10-ylium isomer (CMPQ). The second protonation event closely correlates with the pKa of the N1 nitrogen of C5-protonated dihydro-NAM and fluorophore stability. The fusion partner of oABA is likely the iminium of the primary acid product of dihydro-NAM after glycosidic bond hydrolysis and before irreversible cyclization. Trapping of protonated dihydro-NAM from NADH or NADPH with oABA allows quantification of these dinucleotides. Despite almost a century of research studying acid-catalyzed molecular rearrangements of NADH and NADPH, new and surprising details can be discovered.

EGF Induces Migration Independent of EMT or Invasion in A549 Lung Adenocarcinoma Cells.

Tumors and the tumor microenvironment produce multiple growth factors that influence cancer cell behavior via various signal transduction pathways. Growth factors, like transforming growth factor ß (TGFß) and epidermal growth factor (EGF), have been shown to induce proliferation, migration, and invasion in different cell models. Both factors are frequently overexpressed in cancer and will often act in combination. Although both factors are being used as rational targets in clinical oncology, the similarities and differences of their contributions to cancer cell migration and invasion are not fully understood. Here we compared the impact of treating A549 lung adenocarcinoma cells with TGFß, EGF, and both in combination by applying videomicroscopy, functional assays, immunoblotting, real-time PCR, and proteomics. Treatment with both factors stimulated A549 migration to a similar extent, but with different kinetics. The combination had an additive effect. EGF-induced migration depended on activation of the mitogen-activated protein kinase (MAPK) pathway. However, this pathway was dispensable for TGFß-induced migration, despite a strong activation of this pathway by TGFß. Proteome analysis (data are available via ProteomeXchange with identifier PXD023024) revealed an overlap in expression patterns of migration-related proteins and associated gene ontology (GO) terms by TGFß and EGF. Further, only TGFß induced the expression of epithelial to mesenchymal transition (EMT)-related proteins like matrix metalloproteinase 2 (MMP2). EGF, in contrast, made no major contribution to EMT marker expression on either the protein or the transcript level. In line with these expression patterns, TGFß treatment significantly increased the invasive capacity of A549 cells, while EGF treatment did not. Moreover, the addition of EGF failed to enhance TGFß-induced invasion. Overall, these data suggest that TGFß and EGF can partly compensate for each other for stimulation of cell migration, but abrogation of TGFß signaling may be more suitable to suppress cell invasion.

Characteristics and outcomes of fetuses with laterality defects are the current outcomes better? A single center study.

Objective: To analyze the more recent outcomes of fetuses with different laterality defects.Material and methods: Out of 1508 fetuses with congenital heart disease (CHD) diagnosed by echocardiography between January 1995 and December 2015, 94 (6.3%) presented abnormal visceroatrial situs: 42 till December 2002 (group 1, analyzed retrospectively) and 52 since then until December 2015 (group 2, analyzed prospectively). Twenty-one had left isomerism (Lisom), 22 right isomerism (Risom), 16 situs inversus- dextrocardia (SVI-dx), 5 situs inversus- levocardia (SVI-levo), and 30 situs solitus-dextrocardia (SSol-dx).Results: Seventy-six cases (81%) had CHD, more frequently complex atrioventricular defect. Eighteen cases showed normal heart: half of subgroups SVI-dx and SVI-levo, a third of SSol-dx. Postoperative mortality in continuing pregnancy in group 2 was 43.7 versus 40% in group 1, lower in SVs-dx (0 versus 50%) and Lisom (33.3 versus 66.7%), worse in Risom (71.4 versus 25%). Total mortality in group 2 was 48.3 versus 55% in group 1, better in Ssol-dx (37.5 versus 50%) and in Lisom (27.4 versus 71.4%). In isolated forms all but one case are alive.Conclusions: Our fetal cases presented a relevant mortality in both periods. The outcomes were slightly better in a more recent era in SSol and Lisom.What is known about this topic?Out of laterality defects, the heterotaxies (i.e. left and right isomerism) are known to be associated to a relevant mortality in fetal and neonatal cases.Heterotaxies have a variable position of the heart, but some authors evaluated only those with dextrocardia in their studies on different laterality defects.Controversial data are presented in the literature for the outcomes of fetuses with laterality defects. Many large studies analyzed the data of a very long time period in which it is opinable to compare the results of the treatment, because of a recent improved management and surgical techniques.What does this study add?Our study presents an experience of a single center with cases of different types of laterality defects observed during a routine fetal echocardiography, not only heterotaxies.The knowledge of different laterality defects and of their characteristics (association with cardiac and extracardiac anomalies or with normal heart) is useful for the prenatal counseling.We compare the postoperative and total mortality in more recent period (since 2003) with respect to the previous era (1995-2002).

Expression of FGFR1-4 in Malignant Pleural Mesothelioma Tissue and Corresponding Cell Lines and its Relationship to Patient Survival and FGFR Inhibitor Sensitivity.

Malignant pleural mesothelioma (MPM) is a devastating malignancy with limited therapeutic options. Fibroblast growth factor receptors (FGFR) and their ligands were shown to contribute to MPM aggressiveness and it was suggested that subgroups of MPM patients could benefit from FGFR-targeted inhibitors. In the current investigation, we determined the expression of all four FGFRs (FGFR1-FGFR4) by immunohistochemistry in tissue samples from 94 MPM patients. From 13 of these patients, we were able to establish stable cell lines, which were subjected to FGFR1-4 staining, transcript analysis by quantitative RT-PCR, and treatment with the FGFR inhibitor infigratinib. While FGFR1 and FGFR2 were widely expressed in MPM tissue and cell lines, FGFR3 and FGFR4 showed more restricted expression. FGFR1 and FGFR2 showed no correlation with clinicopathologic data or patient survival, but presence of FGFR3 in 42% and of FGFR4 in 7% of patients correlated with shorter overall survival. Immunostaining in cell lines was more homogenous than in the corresponding tissue samples. Neither transcript nor protein expression of FGFR1-4 correlated with response to infigratinib treatment in MPM cell lines. We conclude that FGFR3 and FGFR4, but not FGFR1 or FGFR2, have prognostic significance in MPM and that FGFR expression is not sufficient to predict FGFR inhibitor response in MPM cell lines.

FGF5 is expressed in melanoma and enhances malignancy in vitro and in vivo.

Although FGF5 mRNA was previously found expressed in some melanoma cell lines in contrast to normal human melanocytes, neither its contribution to melanoma growth nor its expression in melanoma tissue has been investigated. Here we demonstrate that ectopic overexpression of FGF5 in human melanoma cells with low endogenous FGF5 expression increased clonogenicity and invasion but not short-term growth in vitro. Silencing of FGF5 in melanoma cells with high endogenous FGF5 expression had the opposite effect on clonogenicity. FGF overexpression led to increased signaling along the MAPK and NFAT axis but had no effect on STAT3 signaling. In an in vivo experiment in immunocompromised mice, human melanoma xenografts overexpressing FGF5 showed enhanced tumor growth, a higher Ki-67 proliferation index, decreased apoptosis and enhanced angiogenesis. Immunohistochemistry performed on a tissue microarray demonstrated FGF5 protein expression in more than 50% of samples of melanoma and benign nevi. These data suggest that FGF5 has oncogenic potential in melanoma cells and contributes to melanoma growth in a subset of patients. This highlights the importance of further evaluating FGF5 as potential biomarker and therapy target in melanoma.

Left ventricular geometry in normal and post-anterior myocardial infarction patients: sphericity index and 'new' conicity index comparisons.

BACKGROUND: Anterior myocardial infarction leads a sequence of structural changes that alter the size and the shape of the left ventricle. Efforts to assess shape have been made by global left ventricular (LV) chamber analysis (sphericity index, SI) but this analysis does not detect regional shape abnormalities like those at the apical level, which precede global ventricular dilatation. OBJECTIVE: The present study will introduce a new analysis of regional apical changes in 52 normal subjects and in 92 patients with previous anterior myocardial infarction. METHODS: All patients had transthoracic echocardiogram and multiple views were obtained (long axis, 4CH, 2CH and short axis view). From the 4CH view the long and the short axes were measured and their ratio was calculated (sphericity index). In the same view, the apical axis length was also measured and the ratio between apical and short axis length was calculated (apical conicity index, ACI). RESULTS: Patients had all the measured parameters significantly worse than normal, except the sphericity index which remained unchanged. Ventricular length and width increased following anterior MI but the ratio between the two measurements did not change. Conversely, apical conicity index is significantly different following anterior MI, thereby indicating anterior infarction produces a less conical shape. SI and ACI differed when correlations were made in the relationship of mitral valve function; SI correlates with the degree of mitral regurgitation (MR) and with the distance of papillary muscles, conversely ACI shows an inverse correlation with the determinants of mitral regurgitation. These observations reflect differences between apical versus global dilatation in ischemic cardiomyopathy, so that mitral function is better (lower tenting area and lower coaptation height) when the apex is markedly dilated in respect to the short axis (high conicity index). In contrast, mitral function is impaired (bigger distance between papillary muscles and higher degree of mitral regurgitation), when sphericity index is high. CONCLUSIONS: Sphericity index fails to detect regional apical shape abnormalities. To address this focal change, we introduce a simple new measure termed apical conicity index, which is abnormal in patients with myocardial infarction, and can be useful to evaluate changes induced by the subsequent surgical approach of ventricular re-shaping.

Changed outcomes of fetuses with congenital heart disease: new Italian Multicentre study.

OBJECTIVES: To analyse the outcomes of fetuses with congenital heart disease between 2000 and 2005 in comparison to a previous multicentre study regarding the period 1983-1996. PARTICIPANTS AND METHODS: Data of seven centres were prospectively collected, the inclusion criteria being a confirmed fetal diagnosis after birth or at autopsy and a known follow-up, for at least 6 months after birth. Data of 649 fetuses, median age at diagnosis 24 weeks' gestation (15-37), 340/649 (52.4%) diagnosed before 24 weeks, were analysed. RESULTS: Sixty seven and 59 cases had chromosomal or extracardiac anomalies (10.3 and 9.1%). Termination of pregnancy was chosen in 21.6% of cases versus 28.9% in the previous study, being significantly lower in cases with early diagnosis (P <0.001). Out of 509 fetuses continuing pregnancy, 23 died in utero (4.5%) and 110 (21.1%) postnatally, versus 43% in the previous study (P <0.0017). Total surgical/postprocedure death occurred in 20.6% (59/287 infants) versus 37% previously (P <0.003), 67 infants being premature and 35 with associated chromosomal or extracardiac anomalies. The current overall mortality rate was higher in cases with chromosomal or extracardiac anomalies (59.5 and 51.35%, respectively, whereas it was 20.1% in isolated congenital heart disease). Overall current survival was 376 of 509 (73.9%) versus 45% in the previous study (P <0.0001). CONCLUSION: Our data show a reduced overall and surgical mortality, with respect to our previous study, resulting from resulting from an improved perinatal management and treatment of affected fetuses in the more recent era.

Recurrence of congenital heart disease in cases with familial risk screened prenatally by echocardiography.

OBJECTIVES: To evaluate the recurrence of congenital heart disease (CHD) in pregnant women with familial risk who had been referred for fetal echocardiography. MATERIAL AND METHODS: 1634 pregnancies from 1483 women with familial history of CHD in one or more relatives were studied. Fetal cardiologic diagnosis was compared with postnatal findings at 6 months or at autopsy. RESULTS: Total recurrence rate of CHD was 3.98%, 4.06% in single familial risk, 2.9% in double, and 5% in multiple risk. It was 3.5% in case of one previously affected child; 4.5% with 2 children; 5.2% with the mother alone affected and 7,5% with father alone affected and 3.5% with a single distant relative. Exact concordance of CHD was found in 21.5% and a partial concordance in 20% of cases. CONCLUSIONS: Our data show a higher recurrence rate of CHD than previously published data and high relative risk ratios compared to normal population.

Massive myocardial infarction in a full-term newborn: a case report.

A full-term female newborn with neonatal asphyxia and severe anemia (Hb 2.5 g/dL) with normal heart developed a massive myocardial infarction. No examinations were performed during pregnancy for parental nomadism. The baby had immediate external cardiac massage, ventilatory assistance, and blood transfusion. Cardiomegaly was evident at chest X-ray and marked signs of ischemia-lesion at ECG. Echocardiography showed dilated, hypertrophic, and hypocontractile left ventricle (LV), mitral and tricuspid regurgitation, and moderate pericardial effusion. Rh isoimmunization and infective agents were excluded at laboratory tests. Despite the treatment with inotropes, hydrocortisone, and furosemide, the baby worsened and died at 45 hours of life. Postmortem examination showed diffuse subendocardial infarction of LV and diffuse parenchymal hemorrhages and myocardial hypertrophy, increase of eosinophilia, and polymorphonucleated cells at histology. Our patient suffered apparently from longstanding fetal anemia of unknown etiology that led to perinatal distress, severe hypoxia, and massive myocardial infarction, unresponsive to the therapy.

Features and outcomes in utero and after birth of fetuses with myocardial disease.

Objectives. Ninety-one fetuses with dilated or hypertrophic cardiomyopathy (DCM, HCM) and myocarditis were studied. Results. Group 1 "DCM" included 19 fetuses: 13 with hydrops (FH) and 5 with associated extracardiac anomalies (ECAs) (15.8%). Group 2 "Myocarditis" included twelve fetuses, having 11 with FH. Group 3 "HCM" included sixty fetuses: 26 had associated ECAs, 17 had maternal diabetes, and 17 were "idiopathic"; however, in one case, a metabolic disorder was found postnatally, and 4 had familiarity for HCM. Outcomes. Ten cases opted for termination of pregnancy. Two cases with DCM and 1 with HCM were lost at follow-up. Out of the cases that continued pregnancy, with known follow-up, mortality was 68.75% in Group 1, 63.6% in Group 2, and 31.3% in Group 3 (the majority with severe ECAs). Surviving cases with DCM and myocarditis improved, 2 with HCM worsened, 6 remained stable, and 26 improved or normalized. Conclusions. Our data show more severe prognosis in DCM and myocarditis and forms with severe associated ECAs.

Effectiveness of surgical ventricular restoration in patients with dilated ischemic cardiomyopathy and unrepaired mild mitral regurgitation.

OBJECTIVE: Any grade of ischemic mitral regurgitation is associated with excess mortality. Whether mild ischemic mitral regurgitation should be repaired at the time of either coronary artery bypass grafting or surgical ventricular restoration is controversial. Surgical ventricular restoration is a treatment option for dilated post-infarction cardiomyopathy and has the potential to improve mitral functioning. The present study assessed the effectiveness of surgical ventricular restoration and unrepaired mild ischemic mitral regurgitation on left ventricular geometry, cardiac and functional status, and survival. METHODS: We analyzed 55 patients with previous anterior infarction (age 65 +/- 10 years) and mild chronic functional mitral regurgitation who underwent surgical ventricular restoration and coronary artery bypass grafting without mitral repair at our center. Left ventricular volumes, ejection fraction, and geometric parameters were measured before and after surgery. RESULTS: Even mild ischemic mitral regurgitation is characterized by abnormal left ventricular geometry when compared with that of patients without mitral regurgitation at comparable ventricular volumes and ejection fraction. Surgical ventricular restoration induces a significant decrease in left ventricular volumes, left ventricular diameters, and papillary muscle distance; and an improvement in ejection fraction and New York Heart Association class. Ischemic mitral regurgitation significantly decreases in the majority of patients. Survival is 93% at 1 year and 88% at 3 years. CONCLUSION: Surgical ventricular restoration improves mitral functioning by improving geometry abnormalities. Survival is optimal and greater than would be expected in patients with post-infarction dilated ventricles and depressed left ventricular function. Our data indicate that mitral repair in conjunction with surgical ventricular restoration is unnecessary in such patients.

Surgical therapy for ischemic heart failure: single-center experience with surgical anterior ventricular restoration.

OBJECTIVES: Our objectives were (1) to report operative and long-term mortality in patients submitted to anterior surgical ventricular restoration, (2) to report changes in clinical and cardiac status induced by surgical ventricular restoration, and (3) to report predictors of death in a large cohort of patients operated on at San Donato Hospital, Milan, Italy. METHODS: A total of 1161 consecutive patients (83% men, 62 +/- 10 years) had anterior surgical ventricular restoration with or without coronary artery bypass grafting and with or without mitral repair/replacement. A complete echocardiographic study was performed in 488 of 1161 patients operated on between January 1998 and October 2005 (study group). The indication for surgery was heart failure in 60% of patients, angina, and/or a combination of the two. RESULTS: Thirty-day cardiac mortality was 4.7% (55/1161) in the overall group and 4.9% (24/488) in the study group. Determinants of hospital mortality were mitral valve regurgitation and need for a mitral valve repair/replacement. Mitral regurgitation (>2+) associated with a New York Heart Association class greater than II and with diastolic dysfunction (early-to-late diastolic filling pressure >2) further increases mortality risk. Global systolic function improved postoperatively: ejection fraction improved from 33% +/- 9% to 40% +/- 10% (P < .001); end-diastolic and end-systolic volumes decreased from 211 +/- 73 to 142 +/- 50 and 145 +/- 64 to 88 +/- 40 mL, respectively (P < .001) early after surgery. New York Heart Association functional class improved from 2.7 +/- 0.9 to 1.6 +/- 0.7 (P < .001) late after surgery. Long-term survival in the overall population was 63% at 120 months. CONCLUSIONS: Surgical ventricular restoration for ischemic heart failure reduces ventricular volumes, improves cardiac function and functional status, carries an acceptable operative mortality, and results in good long-term survival. Predictors of operative mortality are mitral regurgitation of 2+ or more, New York Heart Association class greater than II, and diastolic dysfunction (early-to-late diastolic filling pressure >2).