Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.

Phenobarbital for Neonatal Seizures: Response Rate and Predictors of Refractoriness.

Background Phenobarbital is the first-line choice for neonatal seizures treatment, despite a response rate of approximately 45%. Failure to respond to acute anticonvulsants is associated with poor neurodevelopmental outcome, but knowledge on predictors of refractoriness is limited. Objective To quantify response rate to phenobarbital and to establish variables predictive of its lack of efficacy. Methods We retrospectively evaluated newborns with electrographically confirmed neonatal seizures admitted between January 1999 and December 2012 to the neonatal intensive care unit of Parma University Hospital (Italy), excluding neonates with status epilepticus. Response was categorized as complete (cessation of clinical and electrographic seizures after phenobarbital administration), partial (reduction but not cessation of electrographic seizures with the first bolus, response to the second bolus), or absent (no response after the second bolus). Multivariate analysis was used to identify independent predictors of refractoriness. Results Out of 91 newborns receiving phenobarbital, 57 (62.6%) responded completely, 15 (16.5%) partially, and 19 (20.9%) did not respond. Seizure type (p = 0.02), background electroencephalogram (EEG; p ≤ 0.005), and neurologic examination (p ≤ 0.005) correlated with response to phenobarbital. However, EEG (p ≤ 0.02) and seizure type (p ≤ 0.001) were the only independent predictors. Conclusion Our results suggest a prominent role of neurophysiological variables (background EEG and electrographic-only seizure type) in predicting the absence of response to phenobarbital in high-risk newborns.

Children's Headache: Drawings in the Diagnostic Work Up.

OBJECTIVES: This study aims to evaluate the drawings effectiveness in childhood headache assessment. BACKGROUND: Headache is a common cause of pain in children. Although drawings have been used in childhood to recognize psychological insights and pain perception, they were rarely used for headache characterization. METHODS: We collected drawings from 67 subjects with cephalalgia during a 22-month timeframe. The clinical diagnosis was made according to the 2nd edition of The International Headache Classification. Drawings were independently categorized as migraine or tension-type headache (TTH) by two child neuropsychiatrists blinded to the clinical data. Cohen kappa for interrater agreement, sensitivity, specificity, and positive predictive value (PPV) were calculated. Subjects were also divided into three age groups to assess the influence of age. Finally, a control group of 90 subjects was collected and K-means cluster analysis was performed. RESULTS: The drawings had a sensitivity of 85.71 and 81.48%, a specificity of 81.48 and 85.71%, and a PPV of 85.71 and 81.48%, for migraine and TTH diagnosis, respectively. Drawings by the older age group showed the highest predictability degree. Finally, by mean of cluster analysis, 59 of the 67 patients were correctly classified, whereas control subjects were similarly distributed between the two clusters. CONCLUSIONS: Drawings are a useful instrument for migraine and TTH differential diagnosis. Thus, we suggest their inclusion in childhood headache diagnostic assessment.

A painful stiff neck following an ear, nose, and throat surgical procedure: case report.

Grisel syndrome is a rare, nontraumatic atlantoaxial subluxation, typical of developmental ages and characterized by head flexion/rotation and painful fixation. Neurological symptoms may occur. It is secondary to head/neck infections and ear, nose, and throat surgery (adenoidectomy, tonsillectomy, and mastoidectomy). Here, we report the case of a child who presented a painful stiff neck following an adenotonsillectomy, with imaging evidencing an atlantoaxial subluxation. The child showed improvement in his condition following a conservative treatment with antibiotics, anti-inflammatory, and analgesic therapy and cervical collar. We believe it is of great significance for clinicians taking into account this peculiar condition in the differential diagnosis of a stiff neck in pediatric patients, thus avoiding misdiagnosis and delays. Indeed, its diagnosis is mainly based on a focused anamnesis associated with the detection of the typical neuroradiological findings.

Facial emotion recognition in childhood: the effects of febrile seizures in the developing brain.

It has been documented that anteromedial temporal lobe dysfunction can cause impairment in emotional intelligence. In particular, medial temporal lobe epilepsy (MTLE) is associated with disorders in emotion recognition from facial expressions. About one-third of patients with MTLE experienced febrile seizures (FSs) during childhood. In the present study, we investigated facial emotion recognition ability in a group of 38 school-aged children with antecedent FSs and in an age- and sex-matched control group. Children with abnormal general visuoperceptual abilities were excluded. Children with FSs showed lower recognition scores versus controls in both matching (28.64 vs 33.47; p<.0001) and labeling (21.25 vs 23.03; p=.001) facial emotions. Our findings support the hypothesis that FSs can be associated during childhood with a dysfunction within the neural network subserving the processing of facial expressions of the basic emotions.

Pax9 and Gbx2 Interact in the Pharyngeal Endoderm to Control Cardiovascular Development.

The correct formation of the aortic arch arteries depends on a coordinated and regulated gene expression profile within the tissues of the pharyngeal arches. Perturbation of the gene regulatory networks in these tissues results in congenital heart defects affecting the arch arteries and the outflow tract of the heart. Aberrant development of these structures leads to interruption of the aortic arch and double outlet right ventricle, abnormalities that are a leading cause of morbidity in 22q11 Deletion Syndrome (DS) patients. We have recently shown that Pax9 functionally interacts with the 22q11DS gene Tbx1 in the pharyngeal endoderm for 4th pharyngeal arch artery morphogenesis, with double heterozygous mice dying at birth with interrupted aortic arch. Mice lacking Pax9 die perinatally with complex cardiovascular defects and in this study we sought to validate further potential genetic interacting partners of Pax9, focussing on Gbx2 which is down-regulated in the pharyngeal endoderm of Pax9-null embryos. Here, we describe the Gbx2-null cardiovascular phenotype and demonstrate a genetic interaction between Gbx2 and Pax9 in the pharyngeal endoderm during cardiovascular development.

Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells.

In vitro generated mammalian cardiomyocytes provide experimental models for studying normal mammalian cardiomyocyte development, for disease modeling and for drug development. They also promise to inform future therapeutic strategies for repair of injured or diseased myocardium. Here we provide reliable protocols for differentiation of mouse embryonic stem cells into functional cardiomyocytes, together with Notes about trouble shooting and optimizing such protocols for specific cell lines.

Cardiomyocyte Differentiation from Human Embryonic Stem Cells.

In vitro generated human cardiomyocytes hold the ultimate promise for heart patients for repair of injured or diseased myocardium, but they also provide experimental models for studying normal cardiomyocyte development, for disease modeling and for drug development. Here we provide reliable protocols for differentiation of human embryonic stem cells into functional cardiomyocytes, together with Notes about troubleshooting and optimizing such protocols for specific cell lines. This chapter also briefly discusses other published protocols and those further adapted for differentiation of induced pluripotent stem cells into cardiomyocytes.

Theatre Is a Valid Add-On Therapeutic Intervention for Emotional Rehabilitation of Parkinson's Disease Patients.

Conventional medical treatments of Parkinson's disease (PD) are effective on motor disturbances but may have little impact on nonmotor symptoms, especially psychiatric ones. Thus, even when motor symptomatology improves, patients might experience deterioration in their quality of life. We have shown that 3 years of active theatre is a valid complementary intervention for PD as it significantly improves the well-being of patients in comparison to patients undergoing conventional physiotherapy. Our aim was to replicate these findings while improving the efficacy of the treatment. We ran a single-blinded pilot study lasting 15 months on 24 subjects with moderate idiopathic PD. 12 were assigned to a theatre program in which patients underwent "emotional" training. The other 12 underwent group physiotherapy. Patients were evaluated at the beginning and at the end of their treatments, using a battery of eight clinical and five neuropsychological scales. We found that the emotional theatre training improved the emotional well-being of patients, whereas physiotherapy did not. Interestingly, neither of the groups showed improvements in either motor symptoms or cognitive abilities tested by the neuropsychological battery. We confirmed that theatre therapy might be helpful in improving emotional well-being in PD.

Neonatal seizures in preterm newborns: A predictive model for outcome.

BACKGROUND: With a reported prevalence of 22.2%, seizures in preterm newborns represent an emergent challenge, because they are often related to adverse outcome. The electroclinical features of preterm infants with neonatal seizures were evaluated in order to predict outcome. METHODS: From 154 newborns with video-EEG confirmed neonatal seizures admitted to Parma University Hospital between January 1999 and December 2012, we collected 76 preterm newborns with neonatal seizures. Outcome was assessed at least at one year. Student t-test for unpaired data was used to compare means of continuous variables. We applied the χ(2) test to compare nominal data between preterm newborns with favorable versus adverse outcome, and between those with seizures versus those with status epilepticus. Then we determined the independent risk factors for adverse outcome with multivariate logistic regression analysis. RESULTS: Birth weight, Apgar at 1st minute, neurologic examination, EEG, US brain scans and the presence of neonatal status epilepticus were different between preterm newborns with favorable and adverse outcome (p ≤ .049). Furthermore, birth weight, seizure onset, neurologic examination and EEG were different between the group with or without status (p ≤ .031). None of the infants with status epilepticus had a favorable outcome compared to 22.3% of those with neonatal seizures (p = .004). We also identified a predictive model that correctly classified outcome in 85.5% of subjects, with a high sensitivity for adverse outcome (>91.5%). CONCLUSION: The presence of neonatal seizures in preterm newborns is highly related to an adverse outcome that can be predicted since the first days of life.

Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development.

Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

Neonatal status epilepticus: differences between preterm and term newborns.

BACKGROUND: Despite the many studies on neonatal seizures, neonatal status epilepticus (NSE) remains a controversial entity, with no general consensus about its definition. We report the characteristics of newborns with NSE in order to assess whether they showed homogeneous features or displayed clinical and/or instrumental differences depending on gestational age (GA). Preterm and term neonates were compared and risk factors for adverse outcome evaluated. METHODS: From 154 newborns with video-EEG confirmed neonatal seizures admitted to the NICU of Parma University Hospital between January 1999 and December 2012, we collected a cohort of 47 newborns (19 preterm, 28 full-term) with NSE. NSE was defined as continuous seizure activity for at least 30 min or recurrent seizures lasting a total of 30 min without definite return to the baseline neurologic condition between seizures. Outcome was assessed at least at one year. We applied the χ(2) test to compare nominal data, and multivariate logistic regression analysis to determine independent risk factors for adverse outcome. RESULTS: Only Apgar scores and neurologic examination (p ≤ .02) were different between the groups. None of the preterm newborns had a favourable outcome compared to 25% of the full-term ones (p = .032). Moreover, 52.6% of preterm neonates died compared to 17.8% of the full-term newborns (p = .01; OR = 5.11). The only variable related to outcome was Apgar score at 5 min (p = .02). CONCLUSION: Newborns with NSE represented a quite homogeneous group regardless of the GA. Outcome was unfavourable in most of the subjects; however adverse outcome and death were more represented in preterm newborns.

RhoA controls Wnt upregulation on microstructured titanium surfaces.

Rough topography enhances the activation of Wnt canonical signaling in vitro, and this mediates its effects on cell differentiation. However, the molecular mechanisms underlying topography-dependent control of Wnt signaling are still poorly understood. As the small GTPase RhoA controls cytoskeletal reorganization and actomyosin-induced tensional forces, we hypothesized that RhoA could affect the activation of Wnt signaling in cells on micropatterned titanium surfaces. G-LISA assay revealed that RhoA activation was higher in C2C12 cells on rough (SLA) surfaces under basal conditions than on smooth (Polished) titanium. Transfection with dominant negative RhoA decreased Wnt activation by normalized TCF-Luc activity on SLA, whilst transfection with constitutively active RhoA increased TCF-Luc activation on Polished titanium. One mM Myosin II inhibitor Blebbistatin increased RhoA activation but decreased Wnt activation on SLA surfaces, indicating that tension-generating structures are required for canonical Wnt modulation on titanium surfaces. Actin inhibitor Cytochalasin markedly enhanced RhoA and TCF-Luc activation on both surfaces and increased the expression of differentiation markers in murine osteoblastic MC3T3 cells. Taken together, these data show that RhoA is upregulated in cells on rough surfaces and it affects the activation of Wnt canonical signaling through Myosin II modulation.