Filament Negative Regulator CDC4 Suppresses Glycogen Phosphorylase Encoded GPH1 That Impacts the Cell Wall-Associated Features in Candida albicans.

We have previously identified Candida albicans GPH1 (orf19.7021) whose protein product was associated with C. albicans Cdc4. The GPH1 gene is a putative glycogen phosphorylase because its Saccharomyces cerevisiae homolog participates in glycogen catabolism, which involves the synthesis of ß-glucan of the fungal cell wall. We made a strain whose CaCDC4 expression is repressed, and GPH1 is constitutively expressed. We established a GPH1 null mutant strain and used it to conduct the in vitro virulence assays that detect cell wall function. The in vitro virulence assay is centered on biofilm formation in which analytic procedures are implemented to evaluate cell surface hydrophobicity; competence, either in stress resistance, germ tube formation, or fibronection association; and the XTT-based adhesion and biofilm formation. We showed that the constitutively expressed GPH1 partially suppresses filamentation when the CaCDC4 expression is repressed. The C. albicans Gph1 protein is reduced in the presence of CaCdc4 in comparison with the absence of CaCdc4. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant displayed a reduction in the capability to form germ tubes and the cell surface hydrophobicity but an increase in binding with fibronectin. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant showed a rise in adhesion, the initial stage of biofilm formation, but displayed a similar capacity to form a mature biofilm. There was no major impact on the gph1Δ/gph1Δ mutant regarding the conditions of cell wall damaging and TOR pathway-associated nutrient depletion. We conclude that GPH1, adversely regulated by the filament suppressor CDC4, contributes to cell wall function in C. albicans.

THR1 mediates GCN4 and CDC4 to link morphogenesis with nutrient sensing and the stress response in Candida albicans.

Candida albicans (C. albicans) CDC4 (CaCDC4), encoding the F­box protein for the substrate specificity of the Skp1­cullin­F­box E3 ubiquitin ligase complex, suppresses the yeast­to­filament transition in C. albicans. In our previous study, Thr1 was identified as a CaCdc4­associated protein using affinity purification. THR1 encodes a homoserine kinase, which is involved in the threonine biosynthesis pathway. The present study generated a strain with repressible CaCDC4 expression and continuous THR1 expression. Colony and cell morphology analyses, as well as immunoblotting, revealed that the Thr1 protein was detectable under conditions in which the expression of CaCDC4 was repressed and that the filaments resulting from the repressed expression of CaCDC4 were suppressed by the constitutive expression of THR1 in C. albicans. Additionally, by using the CaSAT1­flipper method, the present study produced null mutants of THR1, GCN4, and CaCDC4. The phenotypic consequences were evaluated by growth curves, spotting assays, microscopic analysis, reverse transcription­polymerase chain reaction and XTT­based biofilm formation ability. The results revealed that fewer cells lacking THR1 entered the stationary phase but had no apparent morphological alteration. It was observed that the expression of THR1 was upregulated concurrently with GCN4 during nutrient depletion and that cells lacking GCN4 rescued the lethality of cells in the absence of THR1 in conditions accumulating homoserine in the threonine biosynthesis pathway. Of note, it was found that cells with either CaCDC4 or THR1 loss were sensitive to oxidative stress and osmotic stress, with those with THR1 loss being more sensitive. In addition, it was observed that cells with loss of either CaCDC4 or THR1 exhibited the ability to increase biofilm formation, with those lacking CaCDC4 exhibiting a greater extent of enhancement. It was concluded that CaCDC4 is important in the coordination of morphogenesis, nutrient sensing, and the stress response through THR1 in C. albicans.

Nutrition and Health Survey of Taiwan Elementary School Children 2001-2002: research design, methods and scope.

The "Nutrition and Health Survey of Taiwan's Elementary School Children (2001-2002)" was to investigate the nutritional status, influential dietary and non-dietary factors, health and development, and school performance, as well as the inter-relationships among these factors. The survey adopted a two-staged stratified, clustered probability sampling scheme. Towns and districts in Taiwan with particular ethnic and geographical characteristics were designated into 13 strata including Hakka areas, mountain areas, eastern Taiwan, the Penghu Islands, 3 northern regions, 3 central regions and 3 southern regions. Eight schools were selected from each stratum using the probabilities proportional to sizes method. Twenty-four pupils were randomly selected within each school. The survey included face-to-face interviews and health examinations. Taking seasonal effects into consideration, the face-to-face interviews were evenly allocated into each of the two semesters. A total of 2,419 face-to-face interviews and 2,475 health examinations were completed. Interview data included household information, socio-demographics, 24-hour dietary recall, food frequency, dietary and nutritional knowledge, attitudes and behaviors, physical activity, medical history, oral health, pubertal development, and bone health. Health exam data included anthropometry, blood pressure, physical fitness, bone density, dental health, and blood and urine collection. SUDAAN was used to adjust sampling design effect. There were no significant differences in sibling rank and parental characteristics between respondents and non-respondents, which indicates that our survey is representative and unbiased. The results of this survey will increase our understanding on the nutrition and health status of schoolchildren and can be used to shape public health policy in Taiwan.