Wen Hui Tsai

Loss of the respiratory enzyme citrate synthase directly links the Warburg effect to tumor malignancy

To investigate whether altered energy metabolism induces the Warburg effect and results in tumor malignancy, the respiratory enzyme citrate synthase (CS) was examined, silenced, and the effects analyzed. In human cervical carcinoma cells, RNAi-mediated CS knockdown induced morphological changes characteristic of the epithelial-mesenchymal transition (EMT). This switch accelerated cancer cell metastasis and proliferation in in vitro assays and in vivo tumor xenograft models. Notably, CS knockdown cells exhibited severe defects in respiratory activity and marked decreases in ATP production, but great increases in glycolytic metabolism. This malignant progression was due to activation of EMT-related regulators; altered energy metabolism resulted from deregulation of the p53/TIGAR and SCO2 pathways. This phenotypic change was completely reversed by p53 reactivation via treatment with proteasome inhibitor MG132 or co-knockdown of E3 ligase HDM2 and partially suppressed by ATP treatment. This study directly links the Warburg effect to tumor malignancy via induction of the EMT phenotype.

Long-Term Prognosis of Patients with Infantile-Onset Pompe Disease Diagnosed by Newborn Screening and Treated since Birth

OBJECTIVE To determine the benefit of newborn screening for the long-term prognosis of patients with classic infantile-onset Pompe disease (IOPD). STUDY DESIGN A cohort of patients with classic IOPD were diagnosed by newborn screening, treated with recombinant human acid α-glucosidase (rhGAA), and followed prospectively. Outcome measurements included survival, left ventricular mass, serum creatinine kinase, motor function, mental development, and systemic manifestations. RESULTS Ten patients who presented with left ventricular hypertrophy at diagnosis received rhGAA infusions starting at a median age of 16 days (6-34 days). All patients were cross-reactive immunologic material-positive. After a median treatment time of 63 months (range 28-90 months), all could walk independently, and none required mechanical ventilation. All patients had motor capability sufficient for participating in daily activities, but muscle weakness over the pelvic girdle appeared gradually after 2 years of age. Ptosis was present in one-half of the patients, and speech disorders were common. Anti-rhGAA antibody titers were low (median maximal titer value 1:1600, range: undetectable ∼ 1:12,800). CONCLUSION By studying patients treated since birth who have no significant anti-rhGAA antibody interference, this prospective study demonstrates that the efficacy of rhGAA therapy is high and consistent for the treatment of classic IOPD. This study also exposes limitations of rhGAA treatment. The etiology of the manifestations in these early-treated patients will require further study.

Effects of prefeeding oral stimulation on feeding performance of preterm infants

ObjectiveTo investigate the effects of a prefeeding oral stimulation program on the feeding performance of preterm infants.MethodsA crossover design was used. Nineteen preterm infants who were in the transitional time to full oral feeding served as their own controls. A 5-min oral stimulation program was applied to infants prior to feeding in two of 4 feedings on two consecutive days. Feeding, behavioral state, and physiological parameters of infants in the intervention and control feeding conditions were compared using SPSS software.ResultsThere were two significant findings: (1) Compared to the control condition, infants in the intervention condition achieved a greater intake rate in the initial 5 min of the feeding (P = 0.021). (2) After receiving oral stimulation, a higher percentage of infants moved to the drowsy or quiet alert state from sleep or restlessness before feeding, both on Day 1 (P= 0.016) as well as Day 2 (P = 0.016). No significant differences were found in other feeding parameters, feeding-induced physiological changes (peripheral oxygen saturation levels and pulse rate) and behavioral states between two feeding conditions.ConclusionsOral stimulation had a modulating effect on the prefeeding behavioral states and short-lived beneficial effects on the feeding efficiency of preterm infants.

Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase.

Citrate synthase (CS), the first and rate‐limiting enzyme of the tricarboxylic acid (TCA) cycle, plays a decisive role in regulating energy generation of mitochondrial respiration. Most mitochondrial proteins are synthesized in the cytoplasm as preproteins with an amino (N)‐terminal mitochondrial targeting sequence (MTS) that directs mitochondria‐specific sorting of the preprotein. However, the MTS and targeting mechanism of the human CS protein are not fully characterized. The human CS gene is a single nuclear gene which transcribes into two mRNA variants, isoform a (CSa) and b (CSb), by alternative splicing of exon 2. CSa encodes 466 amino acids, including a putative N‐terminal MTS, while CSb expresses 400 residues with a shorter N terminus, lacking the MTS. Our results indicated that CSa is localized in the mitochondria and the N‐terminal 27 amino acids, including a well‐conserved RXY ↓ (S/A) motif (the RHAS sequence), can efficiently target the enhanced green fluorescent protein (EGFP) into the mitochondria. Furthermore, site‐directed mutagenesis analysis of the conserved basic amino acids and serine/threonine residues revealed that the R9 residue is essential but all serine/threonine residues are dispensable in the mitochondrial targeting function. Moreover, RNA interference (RNAi)‐mediated gene silencing of the preprotein import receptors, including TOM20, TOM22, and TOM70, showed that all three preprotein import receptors are required for transporting CSa into the mitochondria. In conclusion, we have experimentally identified the mitochondrial targeting sequence of human CSa and elucidated its targeting mechanism. These results provide an important basis for the study of mitochondrial dysfunction due to aberrant CSa trafficking. J. Cell. Biochem. 107: 1002–1015, 2009.

Association of Cord Plasma Leptin With Birth Size in Term Newborns

BACKGROUND Leptin is secreted from adipose tissue and plays an important role in obesity. Recent studies have shown that the relationship between Leptin and body fat mass may have ethnic differences. The purpose of our study was to investigate the relationship between venous umbilical cord plasma Leptin and anthropometric markers in term healthy Taiwanese newborns. METHODS Umbilical venous plasma samples were obtained from 98 term neonates (48 males and 50 females) and leptin Levels were analyzed by enzyme-linked immunosorbent assay. RESULTS Umbilical cord plasma Levels of leptin were significantly higher in the female neonates than in males (p<0.001). The large-for-gestationaL age and appropriate-for-gestational age newborns had significantly higher Leptin cord plasma levels than the small-for-gestational age newborns (p<0.01 and p<0.05, respectively). In both male and female neonates, umbilical Leptin Levels showed significant positive correlations with birth weight and birth Length. Multiple Linear regression analysis revealed that birth weight was the only significant predictor of umbilical cord plasma Leptin levels in both male and female neonates. However, the slopes of the regressions between Leptin and birth weight in male and female neonates were not different. CONCLUSION In Taiwanese healthy term neonates, leptin umbilical cord plasma Levels are associated with sex and birth weight of the neonate. The relationship between Leptin and birth weight may differ among different ethnic groups. These findings imply that the relationship between leptin and body fat mass may develop early in life.

Associations among perinatal factors and age of achievement of full oral feeding in very preterm infants.

BACKGROUND Progress to full oral feeding from a tube or parenteral feeding is a complex process for very preterm infants born before 32 weeks of gestation. The influence of infant characteristics and medical complications on feeding progression has not been studied thoroughly. The aim of this study was to constitute a regression model to estimate the postmenstrual age (PMA) of full oral feeding and the length of transition time from the initiation to completion of oral feeding. METHODS A chart review was conducted on very preterm infants born between 2005 and 2010 in one medical center in Taiwan. All enrolled infants were able to take all nutrition by mouth before discharge. RESULTS A total of 117 infants fulfilling the criteria were included. The mean PMAs for the initiation and completion of oral feeding were 33.9 ± 1.7 and 35.1 ± 2.0 weeks, respectively. Infants required 7.5 ± 6.6 days from initiation to full oral feeding. The results of a stepwise regression revealed that the reciprocal of birth weight (beta coefficient = 3.81, p < 0.001), moderate-severe bronchopulmonary dysplasia (beta coefficient = 1.21, p < 0.001), necrotizing enterocolitis (beta coefficient = 0.84, p < 0.005), and patent ductus arteriosus (beta coefficient = 0.69, p < 0.01) were predictors for the PMA of full oral feeding. The regression model incorporating those factors explained 62.5% of the variation in the feeding outcome (p < 0.001). Gender, multiple gestations, mild bronchopulmonary dysplasia, intraventricular hemorrhage, and sepsis had no effect on the feeding outcome. None of the explored factors were significantly correlated with transition time. CONCLUSION A regression model incorporating significant predictors to estimate the PMA of full oral feeding in very preterm infants was suggested. It could enhance communication between health professionals and parents about the feeding progress of infants born very prematurely.

A tightly regulated and reversibly inducible siRNA expression system for conditional RNAi-mediated gene silencing in mammalian cells

RNA interference (RNAi) is a powerful and widely used gene silencing strategy for studying gene function in mammalian cells. Transient or constitutive expression of either small interfering RNA (siRNA) or short hairpin RNA (shRNA) results in temporal or persistent inhibition of gene expression, respectively. A tightly regulated and reversibly inducible RNAi‐mediated gene silencing approach could conditionally control gene expression in a temporal or spatial manner that provides an extremely useful tool for studying gene function involved in cell growth, survival and development.

Cardiac structure and function and effects of enzyme replacement therapy in patients with mucopolysaccharidoses I, II, IVA and VI

BACKGROUND While enzyme replacement therapy (ERT) has been shown to improve endurance and joint mobility for patients with mucopolysaccharidoses (MPS) I, II, IVA and VI, the impact of ERT on cardiac abnormalities remains uncertain. METHODS Medical records and echocardiograms of 28 Taiwanese MPS patients (9 with MPS I, 7 with MPS II, 7 with MPS IVA, and 5 with MPS VI) treated with ERT for 1-10.8years were retrospectively reviewed. RESULTS At start of ERT, z scores>2 were identified in 46% and 75% for left ventricular mass index (LVMI) and interventricular septum thickness in diastole (IVSd) in these patients, respectively. Twenty-four patients (86%) had valvular heart disease. After ERT, the mean IVSd z score of all patients decreased significantly from 3.87 to 2.57 (p=0.016). For 11 patients starting ERT before 12years of age, z scores for both LVMI and IVSd decreased significantly (p<0.01) after ERT. However, the condition of valve regurgitation or stenosis did not show improvement despite ERT. CONCLUSIONS ERT was shown to be an effective therapy for reducing cardiac hypertrophy, with best results seen when ERT was started at an early age. ERT, however, had little impact on valvular heart disease.

Higher prevalence of autism in Taiwanese children born prematurely: A nationwide population-based study

The reported prevalence of autism in preterm and full-term children varies partially because of small sample sizes. Moreover, little is known about the specific factors that contribute to the risk of autism in preterm children. We aimed to compare the prevalence of autism in preterm and full-term children and to identify neonatal risk factors for autism in preterm children using a large national health system database. We analyzed data from 1078 early preterm (<28 weeks of gestation or birth weight<1000 g), 28,947 later preterm (28-36 weeks), and 1,104,071 full-term (≥ 37 weeks) children who were 8-11 years old in 2009. The descending order of prevalence was early preterm (2.2%), later preterm (1.3%), and full-term (0.6%). The prevalence of autism was approximately 2-4 times higher in preterm children than in children born at full-term. The male-female ratio (4:1) in preterm and full-term children was not significantly different. Most of the children were first diagnosed with autism between 3 and 6 years old. Preterm children with autism were not diagnosed earlier than were full-term children. Regression analysis showed that male gender, a very low birth weight, and neonatal cerebral dysfunction were risk factors for autism in the preterm group. We conclude that autism is more prevalent in preterm children. Preventing extremely preterm birth and significant early brain insults may be helpful in reducing the risk of autism in preterm children.

Association between mechanical ventilation and neurodevelopmental disorders in a nationwide cohort of extremely low birth weight infants.

Mechanical ventilation for preterm infants independently contributes to poor neurodevelopmental performance. However, few studies have investigated the association between the duration of mechanical ventilation and the risk for various developmental disorders in extremely low birth weight (ELBW) (<1000g) infants. Using a large nationwide database, we did a 10-year retrospective follow-up study to explore the effect of mechanical ventilation on the incidence of cerebral palsy (CP), autism spectrum disorder (ASD), intellectual disability (ID), and attention-deficit/hyperactivity disorder (ADHD) in ELBW infants born between 1998 and 2001. Seven hundred twenty-eight ELBW infants without diagnoses of brain insults or focal brain lesions in the initial hospital stay were identified and divided into three groups (days on ventilator: ≦2, 3-14, ≧15 days). After adjusting for demographic and medical factors, the infants in the ≧15 days group had higher risks for CP (adjusted hazard ratio: 2.66; 95% confidence interval: 1.50-4.59; p<0.001) and ADHD (adjusted hazard ratio: 1.95; 95% confidence interval: 1.02-3.76; p<0.05), than did infants in the ≦2 days group. The risk for ASD or ID was not significantly different between the three groups. We conclude that mechanical ventilation for ≧15 days increased the risk for CP and ADHD in ELBW infants even without significant neonatal brain damage. Developing a brain-protective respiratory support strategy in response to real-time cerebral hemodynamic and oxygenation changes has the potential to improve neurodevelopmental outcomes in ELBW infants.