Generation of mouse conditional and null alleles of the type III sodium‐dependent phosphate cotransporter PiT‐1

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter‐1 (PiT‐1), was shown to be required. To determine the in vivo role of PiT‐1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT‐1^flox, which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre‐mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT‐1^{Δe3,4/Δe3,4} embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT‐1 mouse alleles have been successfully generated and PiT‐1 has a necessary, nonredundant role in embryonic development. genesis 47:858–863, 2009. © 2009 Wiley‐Liss, Inc.     

From neurodevelopment to neurodegeneration: the interaction of neurofibromin and valosin-containing protein/p97 in regulation of dendritic spine formation

Both Neurofibromatosis type I (NF1) and inclusion body myopathy with Paget''s disease of bone and frontotemporal dementia (IBMPFD) are autosomal dominant genetic disorders. These two diseases are fully penetrant but with high heterogeneity in phenotypes, suggesting the involvement of genetic modifiers in modulating patients'' phenotypes. Although NF1 is recognized as a developmental disorder and IBMPFD is associated with degeneration of multiple tissues, a recent study discovered the direct protein interaction between neurofibromin, the protein product of the NF1 gene, and VCP/p97, encoded by the causative gene of IBMPFD. Both NF1 and VCP/p97 are critical for dendritic spine formation, which provides the cellular mechanism explaining the cognitive deficits and dementia found in patients. Moreover, disruption of the interaction between neurofibromin and VCP impairs dendritic spinogenesis. Neurofibromin likely influences multiple downstream pathways to control dendritic spinogenesis. One is to activate the protein kinase A pathway to initiate dendritic spine formation; another is to regulate the synaptic distribution of VCP and control the activity of VCP in dendritic spinogenesis. Since neurofibromin and VCP/p97 also regulate cell growth and bone metabolism, the understanding of neurofibromin and VCP/p97 in neurons may be applied to study of cancer and bone. Statin treatment rescues the spine defects caused by VCP deficiency, suggesting the potential role of statin in clinical treatment for these two diseases.     

Random forests-based differential analysis of gene sets for gene expression data

In DNA microarray studies, gene-set analysis (GSA) has become the focus of gene expression data analysis. GSA utilizes the gene expression profiles of functionally related gene sets in Gene Ontology (GO) categories or priori-defined biological classes to assess the significance of gene sets associated with clinical outcomes or phenotypes. Many statistical approaches have been proposed to determine whether such functionally related gene sets express differentially (enrichment and/or deletion) in variations of phenotypes. However, little attention has been given to the discriminatory power of gene sets and classification of patients.     

Distinct functional defect of three novel Brugada syndrome related cardiac sodium channel mutations

The Brugada syndrome is characterized by ST segment elevation in the right precodial leads V1-V3 on surface ECG accompanied by episodes of ventricular fibrillation causing syncope or even sudden death. The molecular and cellular mechanisms that lead to Brugada syndrome are not yet completely understood. However, SCN5A is the most well known responsible gene that causes Brugada syndrome. Until now, more than a hundred mutations in SCN5A responsible for Brugada syndrome have been described. Functional studies of some of the mutations have been performed and show that a reduction of human cardiac sodium current accounts for the pathogenesis of Brugada syndrome. Here we reported three novel SCN5A mutations identified in patients with Brugada syndrome in Taiwan (p.I848fs, p.R965C, and p.1876insM). Their electrophysiological properties were altered by patch clamp analysis. The p.I848fs mutant generated no sodium current. The p.R965C and p.1876insM mutants produced channels with steady state inactivation shifted to a more negative potential (9.4 mV and 8.5 mV respectively), and slower recovery from inactivation. Besides, the steady state activation of p.1876insM was altered and was shifted to a more positive potential (7.69 mV). In conclusion, the SCN5A channel defect related to Brugada syndrome might be diverse but all resulted in a decrease of sodium current.     

Unconditional Exact Tests for Equivalence or Noninferiority for Paired Binary Endpoints

Problems of establishing equivalence or noninferiority between two medical diagnostic procedures involve comparisons of the response rates between correlated proportions. When the sample size is small, the asymptotic tests may not be reliable. This article proposes an unconditional exact test procedure to assess equivalence or noninferiority. Two statistics, a sample-based test statistic and a restricted maximum likelihood estimation (RMLE)-based test statistic, to define the rejection region of the exact test are considered. We show the p-value of the proposed unconditional exact tests can be attained at the boundary point of the null hypothesis. Assessment of equivalence is often based on a comparison of the confidence limits with the equivalence limits. We also derive the unconditional exact confidence intervals on the difference of the two proportion means for the two test statistics. A typical data set of comparing two diagnostic procedures is analyzed using the proposed unconditional exact and asymptotic methods. The p-value from the unconditional exact tests is generally larger than the p-value from the asymptotic tests. In other words, an exact confidence interval is generally wider than the confidence interval obtained from an asymptotic test.     

Tea Consumption and Risk of Head and Neck Cancer

Background

The current study evaluated the association between tea consumption and head and neck cancer (HNC) in Taiwan, where tea is a major agricultural product and a popular beverage.

Methods

Interviews regarding tea consumption (frequency, duration, and types) were conducted with 396 HNC cases and 413 controls. Unconditional logistic regression was performed to estimate the odds ratio (OR) and 95% confidence interval (CI) of HNC risk associated with tea drinking, adjusted for sex, age, education, cigarette smoking, betel quid chewing, and alcohol drinking.

Results

A reduced HNC risk associated with tea drinking (OR for every cup per day = 0.96, 95% CI: 0.93–0.99; OR for ≧5 cups per day = 0.60, 95% CI: 0.39–0.94) was observed. The association was especially significant for pharyngeal cancer (OR for every cup per day = 0.93, 95% CI: 0.88–0.98; OR for ≧5 cups per day = 0.32, 95% CI: 0.16–0.66). A significant inverse association between HNC and tea consumption was observed particularly for green tea.

Conclusions

This study suggests that tea drinking may reduce the risk of HNC. The anticancer property of tea, if proven, may offer a natural chemopreventive measure to reduce the occurrence of HNC.     

Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles

In addition to pituitary gonadotropins and paracrine factors, ovarian follicle development is also modulated by oocyte factors capable of stimulating granulosa cell proliferation but suppressing their differentiation. The nature of these oocyte factors is unclear. Because growth differentiation factor-9 (GDF-9) enhanced preantral follicle growth and was detected in the oocytes of early antral and preovulatory follicles, we hypothesized that this oocyte hormone could regulate the proliferation and differentiation of granulosa cells from these advanced follicles. Treatment with recombinant GDF-9, but not FSH, stimulated thymidine incorporation into cultured granulosa cells from both early antral and preovulatory follicles, accompanied by increases in granulosa cell number. Although GDF-9 treatment alone stimulated basal steroidogenesis in granulosa cells, cotreatment with GDF-9 suppressed FSH-stimulated progesterone and estradiol production. In addition, GDF-9 cotreatment attentuated FSH-induced LH receptor formation. The inhibitory effects of GDF-9 on FSH-induced granulosa cell differentiation were accompanied by decreases in the FSH-induced cAMP production. These data suggested that GDF-9 is a proliferation factor for granulosa cells from early antral and preovulatory follicles but suppresses FSH-induced differentiation of the same cells. Thus, oocyte-derived GDF-9 could account, at least partially, for the oocyte factor(s) previously reported to control cumulus and granulosa cell differentiation.     

Association of hepatitis virus infection,alcohol consumption and plasma vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical workers

Urinary 8-hydroxydeoxyguanosine (8-OHdG) DNA adduct has been used as a biomarker in epidemiological studies. However, the determinants for urinary 8-OHdG have not been clearly identified. We tested urinary 8-OHdG levels in 205 male workers who had been exposed to vinyl chloride monomer (VCM). Epidemiological information was obtained by an interviewer-administered questionnaire. Hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV) were also determined by immunoassay. Plasma antioxidants including Vitamins A and E, alpha- and beta-carotenes were assayed by high performance liquid chromatography. Median of urinary 8-OHdG level was 9.8 ng/mg creatinine (range, 1.4-60.1). Multiple linear regression analysis showed that alcohol drinkers had higher urinary 8-OHdG than those who did not, but there was no dose-response between the amount of alcohol consumption and urinary 8-OHdG. Workers with positive HBsAg, anti-HCV and elevated plasma Vitamin A level were independently associated with higher levels of urinary 8-OHdG, whereas age, smoking, body mass index, plasma alpha- and beta-carotenes, Vitamin E levels, or VCM exposure did not show such an association. The results suggest that active inflammation of hepatitis B and C, alcohol consumption and higher Vitamin A level can induce oxidative stress. Thus, we conclude that potential determinants need to be considered in epidemiological studies when urinary 8-OHdG is used as a biomarker.     

Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp.

The endosymbiotic relationship between cnidarians and Symbiodinium is critical for the survival of coral reefs. In this study, we developed a protocol to rapidly and freshly separate Symbiodinium from corals and sea anemones. Furthermore, we compared these freshly‐isolated Symbiodinium with cultured Symbiodinium to investigate host and Symbiodinium interaction. Clade B Symbiodinium had higher starch content and lower lipid content than those of clades C and D in both freshly isolated and cultured forms. Clade C had the highest lipid content, particularly when associated with corals. Moreover, the coral‐associated Symbiodinium had higher protein content than did cultured and sea anemone‐associated Symbiodinium. Regarding fatty acid composition, cultured Symbiodinium and clades B, C, and D shared similar patterns, whereas sea anemone‐associated Symbiodinium had a distinct pattern compared coral‐associated Symbiodinium. Specifically, the levels of monounsaturated fatty acids were lower than those of the saturated fatty acids, and the level of polyunsaturated fatty acids (PUFAs) were the highest in all examined Symbiodinium. Furthermore, PUFAs levels were higher in coral‐associated Symbiodinium than in cultured Symbiodinium. These results altogether indicated that different Symbiodinium clades used different energy storage strategies, which might be modified by hosts.