Sylviane Dewaele

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Wenonah Vercoutere, Stephen Winters-Hilt, Hugh Olsen, David Deamer, David Haussler, and Mark Akeson Nat. Biotechnol. 19, 248–252 (2001). The URL given for the DNA mfold server in Table 1 (p. 249 ) and in the text (p. 251) is incorrect. The correct URL is http://bioinfo.math.rpi.edu/~mfold/dna/form1.cgiConverting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

N-glycomic changes in hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus†

We evaluated the use of blood serum N‐glycan fingerprinting as a tool for the diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis induced by hepatitis B virus (HBV). A group of 450 HBV‐infected patients with liver fibrosis or cirrhosis with or without HCC were studied. HCC was diagnosed by α‐fetoprotein (AFP) analysis, ultrasonography, and/or computed tomography and was studied histologically. N‐glycan profiles of serum proteins were determined with DNA sequencer–based carbohydrate analytical profiling technology. In this study, we found that a branch alpha(1,3)‐fucosylated triantennary glycan was more abundant in patients with HCC than in patients with cirrhosis, patients with fibrosis, and healthy blood donors, whereas a bisecting core alpha(1,6)‐fucosylated biantennary glycan was elevated in patients with cirrhosis. The concentration of these 2 glycans and the log ratio of peak 9 to peak 7 (renamed the GlycoHCCTest) were associated with the tumor stage. Moreover, for screening patients with HCC from patients with cirrhosis, the overall sensitivity and specificity of the GlycoHCCTest were very similar to those of AFP. Conclusion: This study indicates that a branch alpha(1,3)‐fucosylated glycan is associated with the development of HCC. The serum N‐glycan profile is a promising noninvasive method for detecting HCC in patients with cirrhosis and could be a valuable supplement to AFP in the diagnosis of HCC in HBV‐infected patients with liver cirrhosis. Its use for the screening, follow‐up, and management of patients with cirrhosis and HCC should be evaluated further. (HEPATOLOGY 2007.)

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-α-d-mannosidase

Abstract A cDNA encoding 1,2-α- d -mannosidase mds1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56 266 Da and shows high similarity to the amino acid sequences of 1,2-α- d -mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris . Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae α-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analysed. The enzyme was characterized as a class-I mannosidase.

N-Glycomic Changes in Serum Proteins in Type 2 Diabetes Mellitus Correlate with Complications and with Metabolic Syndrome Parameters

Background Glycosylation, i.e the enzymatic addition of oligosaccharides (or glycans) to proteins and lipids, known as glycosylation, is one of the most common co-/posttranslational modifications of proteins. Many important biological roles of glycoproteins are modulated by N-linked oligosaccharides. As glucose levels can affect the pathways leading to glycosylation of proteins, we investigated whether metabolic syndrome (MS) and type 2 diabetes mellitus (T2DM), pathological conditions characterized by altered glucose levels, are associated with specific modifications in serum N-glycome. Methods We enrolled in the study 562 patients with Type 2 Diabetes Mellitus (T2DM) (mean age 65.6±8.2 years) and 599 healthy control subjects (CTRs) (mean age, 58.5±12.4 years). N-glycome was evaluated in serum glycoproteins. Results We found significant changes in N-glycan composition in the sera of T2DM patients. In particular, α(1,6)-linked arm monogalactosylated, core-fucosylated diantennary N-glycans (NG1(6)A2F) were significantly reduced in T2DM compared with CTR subjects. Importantly, they were equally reduced in diabetic patients with and without complications (P<0.001) compared with CTRs. Macro vascular-complications were found to be related with decreased levels of NG1(6)A2F. In addition, NG1(6)A2F and NG1(3)A2F, identifying, respectively, monogalactosylated N-glycans with α(1,6)- and α(1,3)-antennary galactosylation, resulted strongly correlated with most MS parameters. The plasmatic levels of these two glycans were lower in T2DM as compared to healthy controls, and even lower in patients with complications and MS, that is the extreme “unhealthy” phenotype (T2DM+ with MS). Conclusions Imbalance of glycosyltransferases, glycosidases and sugar nucleotide donor levels is able to cause the structural changes evidenced by our findings. Serum N-glycan profiles are thus sensitive to the presence of diabetes and MS. Serum N-glycan levels could therefore provide a non-invasive alternative marker for T2DM and MS.

A high-throughput screening system for genes extending life-span.

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging life-span in the bakers yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with extended number of cell divisions as indicated by the increased number of bud scars on their surface. Fluorescently labelled Wheat Germ Agglutinin was used for specific staining of chitin, a major component of bud scars. Screening of a human HepG2 cDNA expression library in yeast resulted in the isolation of 12 yeast transformants with a potentially prolonged life-span. The transgene in one of the lines was identified as ferritin light chain (FTL) and studied in more detail. Yeast cells containing FTL showed an enhanced iron and H(2)O(2) resistance, a reduced cell death rate and an increased number of cell divisions. Overexpression of FTL in the nematode Caenorhabditis elegans resulted in a life-span increase of 8% confirming our yeast observations in a multicellular organism. Our data demonstrate that this method permits a fast screening of libraries for hunting genes involved in ageing processes.

Expression of the human ferritin light chain in a frataxin mutant yeast affects ageing and cell death

Ferritin is one of the major eukaryotic proteins involved in regulating iron metabolism and maintaining iron homeostasis. However, Saccaromyces cerevisiae is an exception, possessing no ferritin and using other means to store excess iron. The only potential iron storage protein identified in yeast so far is the homologue of human frataxin (YFH1p). In this study, we found that dysfunction of yeast frataxin shortens mean lifespan by 49% compared to the WT control. Interestingly, the human ferritin L gene can, at least partially, complement the function of yeast frataxin, extending lifespan and protecting cells from death induced by oxidative stress or excess iron. Our findings indicate that ferritin L can perform functions in yeast that are similar to its functions in mammals, and suggest that common mechanisms may exist for preventing iron and oxidative damage in single- and multi-cellular eukaryotic organisms. Clearly, elucidation of the function of human ferritin in yeast would help in gaining a better understanding the molecular basis of iron storage diseases.

Alteration in N‐glycomics during mouse aging: a role for FUT8

We recently reported that N‐glycosylation changes during human aging. To further investigate the molecular basis determining these alterations, the aging process in mice was studied. N‐glycan profiling of mouse serum glycoproteins in different age groups of healthy C57BL/6 mice showed substantial age‐related changes in three major N‐glycan structures: under‐galactosylated biantennary (NGA2F), biantennary (NA2), and core α‐1,6‐fucosylated ‐β‐galactosylated biantennary structures (NA2F). Mice defective in klotho gene expression (kl/kl), which have a shortened lifespan, displayed a similar but accelerated trend. Interestingly, the opposite trend was observed in slow‐aging Snell Dwarf mice (dw/dw) and in mice fed a calorically restricted diet. We also discovered that increased expression and activity of α‐1,6‐fucosyltransferase (FUT8) in the liver are strongly linked to the age‐related changes in glycosylation and that this increased FUT8 and fucosylation influence IGF‐1 signaling. These data demonstrate that the glycosylation machinery in liver cells is significantly affected during aging and that age‐related increased FUT8 activity could influence the aging process by altering the sensitivity of the IGF‐1R signaling pathway.

Altered serum glycomics in Alzheimer disease: a potential blood biomarker?

We investigated whether blood N-glycan changes can be used as a diagnostic biomarker for Alzheimer disease (AD). We used DNA sequencer-assisted, fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) technology to assay N-glycans in sera from 79 autopsy-confirmed dementia patients and 149 healthy controls. One N-glycan (NA2F) was substantially decreased in AD patients but not in controls. Use of NA2F for discriminating AD between dementia patients and healthy controls showed a diagnostic accuracy of 85.7% +/- 2.8% with 92% specificity and 70% sensitivity. The decrease in the level of NA2F in AD patients compared to non-AD patients was more pronounced in females (p < 0.0001) than in males (p < 0.014). Use of NA2F to differentiate female AD from female non-AD patients reached a diagnostic accuracy of 90.7% +/- 4.8 %. Pearson correlation analysis showed that in female dementia patients, serum NA2F levels were significantly correlated with the cerebrospinal fluid (CSF) beta-amyloid peptide of 42 amino acids (Abeta(1-42)) and tau phosphorylated at threonine 181 (P-tau(181P)) levels, whereas in male dementia patients serum NA2F levels were significantly correlated only with CSF total tau protein (T-tau) level. Thus, we suggest that the serum N-glycan marker might be suitable for longitudinal and follow-up studies.

Altered serum N-glycomics in chronic hepatitis B patients

Background: We previously reported on serum N‐glycans as markers for the diagnosis of cirrhosis in patients with chronic hepatitis C infection. Our present study aimed to evaluate the use of serum glycan markers for the diagnosis of liver fibrosis in patients with chronic hepatitis B infection.

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat

BackgroundThere is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model.ResultsRat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. Conclusions: We found an increase in core-α-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.