Increased demand for FAD synthesis in differentiated and stem pancreatic cancer cells is accomplished by modulating FLAD1 gene expression: the inhibitory effect of Chicago Sky Blue.

FLAD1, along with its FAD synthase (FADS, EC 2.7.7.2) product, is crucial for flavin homeostasis and, due to its role in the mitochondrial respiratory chain and nuclear epigenetics, is closely related to cellular metabolism. Therefore, it is not surprising that it could be correlated with cancer. To our knowledge, no previous study has investigated FLAD1 prognostic significance in pancreatic ductal adenocarcinoma (PDAC). Thus, in the present work, the FAD synthesis process was evaluated in two PDAC cell lines: (a) PANC-1- and PANC-1-derived cancer stem cells (CSCs), presenting the R273H mutation in the oncosuppressor p53, and (b) MiaPaca2 and MiaPaca2-derived CSCs, presenting the R248W mutation in p53. As a control, HPDE cells expressing wt-p53 were used. FADS expression/activity increase was found with malignancy and even more with stemness. An increased FAD synthesis rate in cancer cell lines is presumably demanded by the increase in the FAD-dependent lysine demethylase 1 protein amount as well as by the increased expression levels of the flavoprotein subunit of complex II of the mitochondrial respiratory chain, namely succinate dehydrogenase. With the aim of proposing FADS as a novel target for cancer therapy, the inhibitory effect of Chicago Sky Blue on FADS enzymatic activity was tested on the recombinant 6His-hFADS2 (IC50 = 1.2 µm) and PANC-1-derived CSCs' lysate (IC50 = 2-10 µm). This molecule was found effective in inhibiting the growth of PANC-1 and even more of its derived CSC line, thus assessing its role as a potential chemotherapeutic drug.

Retrograde response to mitochondrial dysfunctions associated to LOF variations in FLAD1 exon 2: unraveling the importance of RFVT2.

Flavin adenine dinucleotide (FAD) synthase (EC 2.7.7.2), encoded by human flavin adenine dinucleotide synthetase 1 (FLAD1), catalyzes the last step of the pathway converting riboflavin (Rf) into FAD. FLAD1 variations were identified as a cause of LSMFLAD (lipid storage myopathy due to FAD synthase deficiency, OMIM #255100), resembling Multiple Acyl-CoA Dehydrogenase Deficiency, sometimes treatable with high doses of Rf; no alternative therapeutic strategies are available. We describe here cell morphological and mitochondrial alterations in dermal fibroblasts derived from a LSMFLAD patient carrying a homozygous truncating FLAD1 variant (c.745C > T) in exon 2. Despite a severe decrease in FAD synthesis rate, the patient had decreased cellular levels of Rf and flavin mononucleotide and responded to Rf treatment. We hypothesized that disturbed flavin homeostasis and Rf-responsiveness could be due to a secondary impairment in the expression of the Rf transporter 2 (RFVT2), encoded by SLC52A2, in the frame of an adaptive retrograde signaling to mitochondrial dysfunction. Interestingly, an antioxidant response element (ARE) is found in the region upstream of the transcriptional start site of SLC52A2. Accordingly, we found that abnormal mitochondrial morphology and impairments in bioenergetics were accompanied by increased cellular reactive oxygen species content and mtDNA oxidative damage. Concomitantly, an active response to mitochondrial stress is suggested by increased levels of PPARγ-co-activator-1α and Peroxiredoxin III. In this scenario, the treatment with high doses of Rf might compensate for the secondary RFVT2 molecular defect, providing a molecular rationale for the Rf responsiveness in patients with loss of function variants in FLAD1 exon 2.HIGHLIGHTSFAD synthase deficiency alters mitochondrial morphology and bioenergetics;FAD synthase deficiency triggers a mitochondrial retrograde response;FAD synthase deficiency evokes nuclear signals that adapt the expression of RFVT2.

Alteration of Flavin Cofactor Homeostasis in Human Neuromuscular Pathologies.

The aim of this short review chapter is to provide a brief summary of the relevance of riboflavin (Rf or vitamin B2) and its derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) for human neuromuscular bioenergetics.Therefore, as a completion of this book we would like to summarize what kind of human pathologies could derive from genetic disturbances of Rf transport, flavin cofactor synthesis and delivery to nascent apoflavoproteins, as well as by alteration of vitamin recycling during protein turnover.

Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy.

Inborn errors of Riboflavin (Rf) transport and metabolism have been recently related to severe human neuromuscular disorders, as resulting in profound alteration of human flavoproteome and, therefore, of cellular bioenergetics. This explains why the interest in studying the "flavin world", a topic which has not been intensively investigated before, has increased much over the last few years. This also prompts basic questions concerning how Rf transporters and FAD (flavin adenine dinucleotide) -forming enzymes work in humans, and how they can create a coordinated network ensuring the maintenance of intracellular flavoproteome. The concept of a coordinated cellular "flavin network", introduced long ago studying humans suffering for Multiple Acyl-CoA Dehydrogenase Deficiency (MADD), has been, later on, addressed in model organisms and more recently in cell models. In the frame of the underlying relevance of a correct supply of Rf in humans and of a better understanding of the molecular rationale of Rf therapy in patients, this review wants to deal with theories and existing experimental models in the aim to potentiate possible therapeutic interventions in Rf-related neuromuscular diseases.

Myocardial performance index and biochemical markers for early detection of doxorubicin-induced cardiotoxicity in children with acute lymphoblastic leukaemia.

BACKGROUND: Despite significant improvements in the prognosis of childhood acute lymphoblastic leukaemia (ALL), the risk of anthracycline-induced cardiovascular disease remains a major concern. This study was designed to investigate the role of the myocardial performance index (MPI) and serum concentrations of biomarkers (cTnT and NT-pro-BNP) in the early detection of subclinical anthracycline-induced functional alterations in children with ALL. METHODS: All children consecutively admitted to our Pediatric Oncologic Department from January 2009 to October 2010 with a diagnosis of ALL were enrolled in this study. cTnT and NT-pro-BNP were evaluated in all patients at diagnosis, before doxorubicin therapy and 2 and 24 h following each anthracycline administration. ECG and echocardiography were performed at diagnosis and 24 h after each anthracycline course. RESULTS: Nineteen children with standard-risk ALL were evaluated. The mean age was 6 years. The cumulative doxorubicin dosage was 240 mg/m(2) according to the AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) ALL 2000 protocol. None of the 19 patients developed congestive heart failure. With increasing cumulative dosages of anthracyclines a significant increase was observed in MPI. This increase was statistically significant starting from the cumulative dosage of 120 mg/m(2) compared to baseline, while the median NT-pro-BNP level did not change significantly during treatment and cTnT levels never exceeded the cut-off value for cardiac injury. CONCLUSION: MPI value is a sensitive and accurate parameter, allowing subclinical cardiac dysfunction to be detected in children receiving anthracyclines. Lifelong cardiac surveillance of these patients is warranted in order to determine the clinical implications of increased MPI on long-term cardiac status.

Kawasaki syndrome and concurrent Coxsackie virus B3 infection.

We describe two previously healthy children who were hospitalized in the same period in different departments of our University with clinical signs of Kawasaki syndrome, which were treated with intravenous immunoglobulins and acetylsalicylic acid: in both cases, Coxsackie virus infection was concurrently demonstrated by enzyme-linked immunosorbent assay, and complement fixation test identified antibodies to serotype B3. In the acute phase, both patients presented hyperechogenic coronary arteries, but no cardiologic sequels in the mid term. The etiological relationship between Kawasaki syndrome and Coxsackie viruses is only hypothetical; however, the eventual identification of ad hoc environmental triggers is advisable in front of children with Kawasaki syndrome, with the aim of optimizing epidemiological surveillance and understanding the intimate biological events of this condition.

Platelet reactivity and endothelial function in children of patients with early acute myocardial infarction.

AIMS: To assess whether platelet reactivity is increased in offspring of patients with early acute myocardial infarction (AMI) and its possible relation with endothelial dysfunction. METHODS AND RESULTS: We studied 23 healthy children (15±3 years, 13 males) of patients with early AMI (≤50 years old; Group 1) and 21 healthy children of healthy subjects without any history of cardiovascular disease (14±3 years, 10 males; Group 2). Platelet reactivity was assessed by flow cytometry as the increase in monocyte-platelet aggregates (MPA) and CD41 and PAC-1 platelet expression in response to exercise stress test (EST), adenosine diphosphate (ADP) stimulation (10(-7) M), or both. Endothelial function was assessed by measuring brachial artery dilation during post-ischaemic forearm hyperaemia [flow-mediated dilation (FMD)]. Both EST and ADP induced a higher percentage increase in platelet receptor expression in Group 1, compared with Group 2, with the most significant difference being shown for the response to the combined stimuli (e.g. MPA, 23.1±12 vs. 5.63±8%, P<0.001; platelet PAC-1, 57.7±47 vs. 13.2±7%, P<0.001). Compared with Group 2, Group 1 children showed lower FMD (10.7±3.1 vs. 8.0±2.9%, respectively; P=0.007). However, no significant association was found between FMD and platelet reactivity. CONCLUSION: Our results show increased platelet reactivity in children of patients with early AMI; the finding was not significantly correlated with endothelial dysfunction, suggesting that other mechanisms are mainly involved in the enhanced platelet response to agonistic stimuli.

Neurogenic stunned myocardium presenting as left ventricular hypertrabeculation in childhood: a variant of Takotsubo cardiomyopathy?

OBJECTIVE: To report the first case of neurogenic stunned myocardium presenting with heart left ventricle noncompaction requiring intensive care in the perioperative period of tension tumor-induced hydrocephalus. METHODS AND DESIGN: Case report and literature review. Our Institutional Review Board waived the need for consent. PATIENT: A 12-yr-old female with intracranial astrocytoma and hypertensive hydrocephalus presented with severe heart dysfunction and life-threatening ventricular ectopies intraoperatively. A severe heart failure developed requiring hemodynamic and ventilatory support for 10 days. Echocardiography showed a transient noncompaction aspect of the left ventricular wall, further confirmed by a cardiac magnetic resonance image. The noncompaction aspect lasted until 15 days postadmission, as was the case for the QT interval prolongation; no life-threatening ectopies were demonstrated on the subsequent Holter electrocardiogram monitoring. CONCLUSIONS: This report describes a unique presentation of myocardial stunning in association with an intracranial illness, namely, a hypertensive hydrocephalus complicating an intracranial neoplasm.

Electrical and autonomic cardiac function in patients with Dravet syndrome.

Dravet syndrome (DS) is an epileptic encephalopathy related mainly to mutations in the SCN1A gene, encoding for neuronal sodium channels. Patients with DS have a high risk of sudden unexpected death in epilepsy (SUDEP). In this study we investigated whether patients with DS present abnormalities in electrical and autonomic cardiac function. To this aim we assessed ventricular repolarization and heart rate variability (HRV) on standard electrocardiography (ECG) and on 24-h ECG Holter monitoring, respectively, in 20 patients affected by DS (6.8 ± 4 years, 11 female). As age- and sex-matched control groups, we also studied 20 patients with other epileptic syndromes receiving antiepileptic drugs (ES/AED, 6.0 ± 5 years, 12 female), 20 patients with other epileptic syndromes without treatment (ES/no-AED, 6.7 ± 4 years, 10 female), and 20 healthy children (HC, 7.2 ± 5 years, 11 females). Data analysis showed that patients with DS had depressed HRV variables compared to both ES patients (ES/AED and ES/no-AED) and HC control group, whereas no significant differences in HRV variables were found between ES patients (with and without treatment) and HC. There was no significant difference between patients with DS and all the other control groups in RR intervals, QT, and QTc interval analysis. In conclusion, DS patients display an imbalance of cardiac autonomic function toward a relative predominance of adrenergic tone compared to both healthy children and patients with other forms of epilepsy, independent of antiepileptic therapy. Follow-up studies should clarify the clinical significance of this autonomic impairment and whether HRV analysis can be helpful in predicting the risk of sudden death in patients with DS.

Exercise-induced rhabdomyolysis and transient loss of deambulation as outset of partial carnitine palmityl transferase II deficiency.

We report the case of a 13-year-old boy with an abrupt onset of leg pain and muscle weakness, incapability of deambulation and a laboratory picture of exercise-induced acute rhabdomyolysis. Intravenous hyperhydration and forced diuresis were adopted to avoid renal complications. No evidence of articular or residual muscular damage was appreciated in the short-term. The recurrence of rhabdomyolysis required a muscular biopsy showing a disturbance of fatty acid ß-oxidation pathway.

Neonatal emergencies associated with cardiac rhabdomyomas: an 8-year experience.

During the foetal-neonatal period, rhabdomyomas represent the majority of cardiac tumours and are closely associated with tuberous sclerosis. Cardiac rhabdomyomas may be completely asymptomatic and are incidentally discovered during an echocardiogram, or may cause cardiac dysfunctions requiring medical and/or surgical intervention. During foetal life and the early neonatal period, life-threatening conditions, mostly due to arrhythmias, cardiac failure or obstruction, do occur on rare occasions. We reviewed the medical records of all cases of cardiac rhabdomyomas diagnosed prenatally or postnatally over an 8-year period. The present study reviews 7 cases of life-threatening conditions. Arrhythmic episodes were described in 5 patients, and blood flow obstruction was reported in 2 cases. Antiarrhythmic agents successfully controlled the clinical and electrophysiological conditions. Obstructive conditions were associated with poor outcomes. In conclusion, when prenatal diagnosis of rhabdomyoma is made, appropriate planning at delivery for the management of potential haemodynamic complications may prevent adverse neonatal outcomes. The clinical outcome is more influenced by obstructive rather than by dysrhythmic complications. Appropriate antiarrhythmic treatment is of primary importance. In all cases discovered through prenatal and/or neonatal life-threatening conditions, an accurate follow-up should always be performed to anticipate the development of tuberous sclerosis.

Isolated pericardial agenesis revealed by bradycardia and heart MRI in a healthy 5-year-old child.

We present a five-year-old boy with an unremarkable medical history who was incidentally found to have bradycardia and electrocardiographic signs of right axial deviation. Initial echocardiogram showed left displacement of the cardiac apex with slight enlargement of the right ventricle, while frontal chest radiograph showed a lucent area between the aorta and pulmonary artery. Cardiac magnetic resonance imaging finally revealed a partial left pericardial agenesis and abnormal displacement of the heart into the left hemithorax.

Occipital porencephaly in a child with gyrate atrophy of the choroid and retina.

A 4-year-old girl was hospitalized for psychomotor delay, low vision, and horizontal nystagmus. She was found to have bilateral chorioretinal atrophic scars and 2 large occipital porencephalic cavities. High plasma ornithine levels led to the presumed diagnosis of gyrate atrophy of the choroid and retina. After 6 months of arginine-restricted diet and high-dose pyridoxine (300 mg/d), there was no change of plasma ornithine level or ocular findings. To our knowledge, this is the first report showing an association of porencephaly with gyrate atrophy of the choroid and retina.

Responsiveness to intravenous immunoglobulins and occurrence of coronary artery abnormalities in a single-center cohort of Italian patients with Kawasaki syndrome.

Clues to predict the response to intravenous immunoglobulins (IVIG) and the development of coronary artery abnormalities (CAA) in children with Kawasaki syndrome (KS) are still undefined. We examined retrospectively the medical charts of children hospitalized between February 1990 and April 2009 with diagnosis of KS. A total of 32 Italian patients with a mean age of 23.8 months were analyzed and all received IVIG according to two schemes: 0.4 g/(kg day) for 5 days or 2 g/kg in a single infusion, combined with oral acetylsalicylic acid. General, clinical and laboratory data were registered. Each patient was evaluated with echocardiography at admission, then with 3-day and weekly frequency, respectively, during hospital stay and for the first 6-8 weeks since onset, and finally with a regular 6-12 month follow-up over time, according to patient risk stratification. Five patients showing significantly higher values of C-reactive protein (CRP) at admission were IVIG-resistant after the first infusion (P = 0.04) in comparison with the remaining 27. Five patients out of 32 developed CAA, with no statistical significance when analyzed for IVIG dosage or IVIG-resistance. The demonstration of CAA was significantly higher in children aged <12 months (P = 0.037). Our experience, limited to a single-center cohort of 32 patients with KS, though treated with two different IVIG schemes, has shown that higher values of CRP and younger age at onset are nodal points in determining, respectively, a failure in the response to IVIG and an increased occurrence of CAA.

Incomplete Kawasaki syndrome followed by systemic onset-juvenile idiopathic arthritis mimicking Kawasaki syndrome.

A 3-month-old child was first treated for incomplete Kawasaki syndrome with three cycles of intravenous immunoglobulins and aspirin, then with methylprednisolone which led to fever remission. The same child was re-hospitalized after a 10-month-period of well-being for the suspicion of a new episode of Kawasaki syndrome, which appeared to be immunoglobulin-resistant: extensive testing failed to provide an alternative diagnosis of any infectious or infiltrative disease. Diagnosis of systemic onset-juvenile idiopathic arthritis was postulated upon the long persistence of fever and inflammatory signs, which subsided only after starting corticosteroid treatment.