👤 A Jong

Atherosclerotic cardiovascular disease, characterized by an imbalanced lipid metabolism and a dysregulated immune response, is a major cause of death worldwide. The AhR (aryl hydrocarbon receptor) is a ligand-activated transcription factor that is highly expressed in the liver and primarily known for its role in detoxification. However, recent studies suggest that the AhR also plays a key role in immune regulation, indicating that this receptor can influence the development of atherosclerosis. The number of circulating leukocytes was increased in Our study demonstrates a remarkable role for AhR in the pathogenesis of atherosclerosis, interfering with both lipid metabolism and inflammatory pathways. Although the underlying mechanisms remain unclear, these results demonstrate a novel and crucial role for AhR in atherosclerotic cardiovascular disease. Show less

Atherosclerosis has an auto-immune component driven by self-reactive T and B cells. Identifying their antigenic drivers may lead to new diagnosis and treatment approaches. Here, we aim to identify immunogenic T cell epitopes derived from atherosclerosis-relevant proteins such as ApoB100 by studying the repertoire of peptides presented by HLA in human plaques. We used immunopeptidomics to identify peptides presented by HLA-DR molecules from plaques of patients that underwent endarterectomy surgery. We selected a set of 20 peptides derived from ApoB100 and studied the presence and cytokine profile of ApoB100-specific CD4 revealed significant CD4 We show that immunopeptidomics can be a valid approach to new discover antigens in atherosclerosis. Show less

Taxonomic analysis of environmental microbial communities is now routinely performed thanks to advances in DNA sequencing. Determining the role of these communities in global biogeochemical cycles requires the identification of their metabolic functions, such as hydrogen oxidation, sulfur reduction, and carbon fixation. These functions can be directly inferred from metagenomics data, but in many environmental applications metabarcoding is still the method of choice. The reconstruction of metabolic functions from metabarcoding data and their integration into coarse-grained representations of biogeochemical cycles remains a difficult bioinformatics problem today. We developed a pipeline, called Tabigecy, which exploits taxonomic affiliations to predict metabolic functions constituting biogeochemical cycles. In a first step, Tabigecy uses the tool EsMeCaTa to predict consensus proteomes from input affiliations. To optimize this process, we generated a precomputed database containing information about 2404 taxa from UniProt. The consensus proteomes are searched using bigecyhmm, a newly developed Python package relying on Hidden Markov Models to identify key enzymes involved in metabolic function of biogeochemical cycles. The metabolic functions are then projected on coarse-grained representation of the cycles. We applied Tabigecy to two salt cavern datasets and validated its predictions with microbial activity and hydrochemistry measurements performed on the samples. The results highlight the utility of the approach to investigate the impact of microbial communities on biogeochemical processes. The Tabigecy pipeline is available at https://github.com/ArnaudBelcour/tabigecy. The Python package bigecyhmm and the precomputed EsMeCaTa database are also separately available at https://github.com/ArnaudBelcour/bigecyhmm and https://doi.org/10.5281/zenodo.13354073, respectively. Show less

Elevated von Willebrand factor (VWF) levels correlate with higher risk of atherosclerosis-related arterial thrombosis (atherothrombosis). Silencing the This study aimed to investigate whether the LNP-siRNA strategy could achieve endothelium-specific Female transgenic mice expressing a variant of human The LNP-siRNA targeting Show less

Colorectal cancer (CRC) is stratified into four consensus molecular subtypes (CMS1-4). CMS3 represents the metabolic subtype, but its wiring remains largely undefined. To identify the underlying tumorigenesis of CMS3, organoids derived from 16 genetically engineered mouse models are analyzed. Upon in vitro Cre-recombinase activation, transformation is established and transcriptional profiling reveals that distinct CMSs (CMS2-4) are modeled with different organoids. CMS3-like, metabolic signature-positive, organoids are induced by KRAS mutations. Interestingly, metabolic signatures are subsequently shown to result from enterocyte-like differentiation both in organoids and human cancers. Further analysis reveals carbamoyl-phosphate synthase 1 (CPS1) and sucrase-isomaltase (SI) as signature proteins. More importantly, CPS1 is crucial for de novo pyrimidine synthesis in CMS3 and its inhibition targets proliferation and stemness, facilitating enterocyte-like differentiation, while CMS2 and CMS4 models are not affected. Our data point to an enterocyte-like differentiation of CMS3 CRCs and reveal a selective vulnerability of this subtype through CPS1 inhibition. Show less

An estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 ( We developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native Our multidimensional mapping strategy enabled high-resolution functional analysis of This work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies. Show less

ChREBP (carbohydrate responsive element binding protein) is a transcription factor that responds to sugar consumption. Sugar-sweetened beverage (SSB) consumption and genetic variants in the Data from 11 cohorts from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium (N=63 599) and the UK Biobank (N=59 220) were used to quantify associations of SSB consumption, genetic variants, and their interaction on HDL-C and triglyceride concentrations using linear regression models. A total of 1606 single nucleotide polymorphisms within or near In a meta-analysis, rs71556729 was significantly associated with higher HDL-C concentrations only among the highest SSB consumers (β, 2.12 [95% CI, 1.16-3.07] mg/dL per allele; Our results identified genetic variants in the Show less

Peanut allergy (PA) is a complex disease with both environmental and genetic risk factors. Previously, PA loci were identified in filaggrin (FLG) and HLA in candidate gene studies, and loci in HLA were identified in a genome-wide association study and meta-analysis. We sought to investigate genetic susceptibility to PA. Eight hundred fifty cases and 926 hyper-control subjects and more than 7.8 million genotyped and imputed single nucleotide polymorphisms (SNPs) were analyzed in a genome-wide association study to identify susceptibility variants for PA in the Canadian population. A meta-analysis of 2 phenotypes (PA and food allergy) was conducted by using 7 studies from the Canadian, American (n = 2), Australian, German, and Dutch (n = 2) populations. An SNP near integrin α6 (ITGA6) reached genome-wide significance with PA (P = 1.80 × 10 This study identifies multiple novel loci as risk factors for PA and food allergy and establishes C11orf30 as a risk locus for both PA and food allergy. Multiple genes (C11orf30/EMSY, SKAP1, and CTNNA3) identified by this study are involved in epigenetic regulation of gene expression. Show less

Sugar-sweetened beverages (SSBs) are a major dietary contributor to fructose intake. A molecular pathway involving the carbohydrate responsive element-binding protein (ChREBP) and the metabolic hormone fibroblast growth factor 21 (FGF21) may influence sugar metabolism and, thereby, contribute to fructose-induced metabolic disease. We hypothesise that common variants in 11 genes involved in fructose metabolism and the ChREBP-FGF21 pathway may interact with SSB intake to exacerbate positive associations between higher SSB intake and glycaemic traits. Data from 11 cohorts (six discovery and five replication) in the CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) Consortium provided association and interaction results from 34,748 adults of European descent. SSB intake (soft drinks, fruit punches, lemonades or other fruit drinks) was derived from food-frequency questionnaires and food diaries. In fixed-effects meta-analyses, we quantified: (1) the associations between SSBs and glycaemic traits (fasting glucose and fasting insulin); and (2) the interactions between SSBs and 18 independent SNPs related to the ChREBP-FGF21 pathway. In our combined meta-analyses of discovery and replication cohorts, after adjustment for age, sex, energy intake, BMI and other dietary covariates, each additional serving of SSB intake was associated with higher fasting glucose (β ± SE 0.014 ± 0.004 [mmol/l], p = 1.5 × 10 In this large meta-analysis, we observed that SSB intake was associated with higher fasting glucose and insulin. Although a suggestive interaction with a genetic variant in the ChREBP-FGF21 pathway was observed in the discovery cohorts, this observation was not confirmed in the replication analysis. Trials related to this study were registered at clinicaltrials.gov as NCT00005131 (Atherosclerosis Risk in Communities), NCT00005133 (Cardiovascular Health Study), NCT00005121 (Framingham Offspring Study), NCT00005487 (Multi-Ethnic Study of Atherosclerosis) and NCT00005152 (Nurses' Health Study). Show less

Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. To analyze the evolution of the buoyant density of in vivo-derived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet. Show less

Fetal akinesia deformation sequence (FADS) refers to a broad spectrum of disorder with the absent fetal movement as the unifying feature. The etiology of FADS is heterogeneous, and the majority remains unknown. Prenatal diagnosis of FADS because of neuromuscular origin has relied on clinical features and fetal muscle pathology, which can be unrevealing. The recent advance of next-generation sequencing (NGS) can provide definitive molecular diagnosis effectively. An 18-week-old fetus presented with akinesia and multiple contractures of joints. The mother had two previously aborted similarly affected fetuses. Clinical diagnosis of FADS was made. Molecular diagnosis using cord blood by NGS of genes related to neuromuscular diseases revealed two compound heterozygous mutations; c.602G > A(p.W201*) and c.1516A > C(p.T506P), in the Kelch-like 40 (KLHL40) gene. Based on this information, prenatal diagnosis was performed on the CVS of the subsequent pregnancy that resulted in an unaffected female baby, heterozygous for the c.1516A > C(p.T506P) mutation. Identification of KLHL40 mutations in one of the aborted fetuses provided a confirmative diagnosis of FADS, facilitating the prenatal diagnosis of the subsequent pregnancy. This report underscores the importance of target NGS in providing FADS families with an affordable, precise molecular diagnosis for genetic counseling and options of prenatal diagnosis. © 2016 John Wiley & Sons, Ltd. Show less

Cryptococcus demonstrates predilection for invasion of the brain, but the mechanism by which Cryptococcus crosses the blood-brain barrier (BBB) to cause brain invasion is largely unknown. In order for Cryptococcus to cross the BBB, there must be a way to either cross human brain microvascular endothelial cells, which are the main constitute of the BBB, or go in between tight junctions. Recent evidence of human brain microvascular endothelial cell responses to transcellular brain invasions includes membrane rearrangements, intracellular signaling pathways and cytoskeletal activations. Several Cryptococcal genes related to the traversal of BBB have been identified, including CPS1, ITR1a, ITR3c, PLB1, MPR1, FNX1 and RUB1. In addition, Cryptococcus neoformans-derived microvesicles may contribute to cryptococcal brain invasion. Paracellularly, Cryptococcus may traverse across BBB using either routes utilizing plasmin, ammonia or macrophages in a Trojan horse mechanism. Show less

Genetic variants in genes encoding components of lipid metabolism have been associated with AMD. The aims of this study were to evaluate the relation of these genetic variants with serum lipid levels in AMD in a large case-control cohort (n = 3070) and to test for correlations between lipids and complement activation. Single nucleotide polymorphisms (SNPs) in eight lipid metabolism genes, previously described to be associated with AMD, were genotyped and tested for their association in our case-control cohort. Serum apolipoprotein B (ApoB), apolipoprotein AI (Apo-AI), cholesterol, triglycerides (TG), high-density lipoprotein-cholesterol (HDLC), and complement activation levels (C3d/C3) were measured and tested for association with AMD. Non-HDL cholesterol and LDL were inferred based on the measurements of the other lipids and lipoproteins. General linear models and χ2 tests were used to evaluate the relation of SNPs and lipids/lipoproteins to the disease as well as their interrelations. Significant genotypic associations with AMD were observed for SNPs in CETP, APOE, and FADS1. The serum levels of Apo-AI and HDLC were significantly higher in patients compared with controls. Triglycerides (TG) levels were lower in AMD compared with controls. A cumulative effect was observed for APOE and CETP genotypes on HDLC and Apo-AI levels. Complement activation levels correlated positively with HDLC and Apo-AI, and negatively with TG. Both the lipids/lipoproteins and the complement activation levels associate independently to AMD. This study bridges the gap between genetic associations and physiological lipid levels in AMD. Additionally, the observed correlations between complement activation and lipid levels link two major systems that previously were always assessed independently. Show less

Forced vital capacity (FVC), a spirometric measure of pulmonary function, reflects lung volume and is used to diagnose and monitor lung diseases. We performed genome-wide association study meta-analysis of FVC in 52,253 individuals from 26 studies and followed up the top associations in 32,917 additional individuals of European ancestry. We found six new regions associated at genome-wide significance (P < 5 × 10(-8)) with FVC in or near EFEMP1, BMP6, MIR129-2-HSD17B12, PRDM11, WWOX and KCNJ2. Two loci previously associated with spirometric measures (GSTCD and PTCH1) were related to FVC. Newly implicated regions were followed up in samples from African-American, Korean, Chinese and Hispanic individuals. We detected transcripts for all six newly implicated genes in human lung tissue. The new loci may inform mechanisms involved in lung development and the pathogenesis of restrictive lung disease. Show less

Multiple osteochondromas (MO) is a syndrome in which benign cartilage-capped neoplasms develop at the surface of the long bones. Most cases are caused by exonic changes in EXT1 or EXT2, but 15% are negative for these changes. Here we report for the first time a family of MO patients with germline genomic alterations at the EXT1 locus without detectable mutations or copy number alterations of EXT exonic sequences. Array-CGH showed an 80.7 kb deletion of Intron 1 of EXT1 and a 68.9 kb duplication proximal of EXT1. We identified a breakpoint between the distal end of the duplicated region and a sequence distal of the deleted region in the first intron. This breakpoint was absent in non-affected family members. The configuration of the breakpoint indicates a direct insertion of the duplicated region into the deletion. However, no other breakpoint was found, which suggests a more complex genomic rearrangement has occurred within the duplicated region. Our results reveal intronic deletion and duplication as a new causative mechanism for MO not detected by conventional diagnostic methods. Show less

Alzheimer's disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD. Show less

Secondary peripheral chondrosarcoma is the result of malignant transformation of a pre-existing osteochondroma, the most common benign bone tumor. Osteochondromas are caused by genetic abnormalities in EXT1 or EXT2: homozygous deletion of EXT1 characterizes sporadic osteochondromas (non-familial/solitary), and germline mutations in EXT1 or EXT2 combined with loss of heterozygosity define hereditary multiple osteochondromas. While cells with homozygous inactivation of EXT and wild-type cells shape osteochondromas, the cellular composition of secondary peripheral chondrosarcomas and the role of EXT in their formation have remained unclear. We report using a targeted-tiling-resolution oligo-array-CGH (array comparative genomic hybridization) that homozygous deletions of EXT1 or EXT2 are much less frequently detected (2/17, 12%) in sporadic secondary peripheral chondrosarcomas than expected based on the assumption that they originate in sporadic osteochondromas, in which homozygous inactivation of EXT1 is found in ~80% of our cases. FISH with an EXT1 probe confirmed that, unlike sporadic osteochondromas, cells from sporadic secondary peripheral chondrosarcomas predominantly retained one (hemizygous deleted loci) or both copies (wild-type) of the EXT1 locus. By immunohistochemistry, we confirm the presence of cells with dysfunctional EXT1 in sporadic osteochondromas and show cells with functional EXT1 in sporadic secondary peripheral chondrosarcomas. These immuno results were verified in osteochondromas and secondary peripheral chondrosarcomas in the setting of hereditary multiple osteochondromas. Our data therefore point to a model of oncogenesis in which the osteochondroma creates a niche in which wild-type cells with functional EXT are predisposed to acquire other mutations giving rise to secondary peripheral chondrosarcoma, indicating that EXT-independent mechanisms are involved in the pathogenesis of secondary peripheral chondrosarcoma. Show less

Sex hormone-binding globulin (SHBG) is a glycoprotein responsible for the transport and biologic availability of sex steroid hormones, primarily testosterone and estradiol. SHBG has been associated with chronic diseases including type 2 diabetes (T2D) and with hormone-sensitive cancers such as breast and prostate cancer. We performed a genome-wide association study (GWAS) meta-analysis of 21,791 individuals from 10 epidemiologic studies and validated these findings in 7,046 individuals in an additional six studies. We identified twelve genomic regions (SNPs) associated with circulating SHBG concentrations. Loci near the identified SNPs included SHBG (rs12150660, 17p13.1, p = 1.8 × 10(-106)), PRMT6 (rs17496332, 1p13.3, p = 1.4 × 10(-11)), GCKR (rs780093, 2p23.3, p = 2.2 × 10(-16)), ZBTB10 (rs440837, 8q21.13, p = 3.4 × 10(-09)), JMJD1C (rs7910927, 10q21.3, p = 6.1 × 10(-35)), SLCO1B1 (rs4149056, 12p12.1, p = 1.9 × 10(-08)), NR2F2 (rs8023580, 15q26.2, p = 8.3 × 10(-12)), ZNF652 (rs2411984, 17q21.32, p = 3.5 × 10(-14)), TDGF3 (rs1573036, Xq22.3, p = 4.1 × 10(-14)), LHCGR (rs10454142, 2p16.3, p = 1.3 × 10(-07)), BAIAP2L1 (rs3779195, 7q21.3, p = 2.7 × 10(-08)), and UGT2B15 (rs293428, 4q13.2, p = 5.5 × 10(-06)). These genes encompass multiple biologic pathways, including hepatic function, lipid metabolism, carbohydrate metabolism and T2D, androgen and estrogen receptor function, epigenetic effects, and the biology of sex steroid hormone-responsive cancers including breast and prostate cancer. We found evidence of sex-differentiated genetic influences on SHBG. In a sex-specific GWAS, the loci 4q13.2-UGT2B15 was significant in men only (men p = 2.5 × 10(-08), women p = 0.66, heterogeneity p = 0.003). Additionally, three loci showed strong sex-differentiated effects: 17p13.1-SHBG and Xq22.3-TDGF3 were stronger in men, whereas 8q21.12-ZBTB10 was stronger in women. Conditional analyses identified additional signals at the SHBG gene that together almost double the proportion of variance explained at the locus. Using an independent study of 1,129 individuals, all SNPs identified in the overall or sex-differentiated or conditional analyses explained ~15.6% and ~8.4% of the genetic variation of SHBG concentrations in men and women, respectively. The evidence for sex-differentiated effects and allelic heterogeneity highlight the importance of considering these features when estimating complex trait variance. Show less

Osteochondromas (cartilage-capped bone tumors) are by far the most commonly treated of all primary benign bone tumors (50%). In 15% of cases, these tumors occur in the context of a hereditary syndrome called multiple osteochondromas (MO), an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped bone tumors at children's metaphyses. MO is caused by various mutations in EXT1 or EXT2, whereby large genomic deletions (single-or multi-exonic) are responsible for up to 8% of MO-cases. Here we report on the first molecular characterization of ten large EXT1- and EXT2-deletions in MO-patients. Deletions were initially identified using MLPA or FISH analysis and were subsequently characterized using an MO-specific tiling path array, allele-specific PCR-amplification and sequencing analysis. Within the set of ten large deletions, the deleted regions ranged from 2.7 to 260 kb. One EXT2 exon 8 deletion was found to be recurrent. All breakpoints were located outside the coding exons of EXT1 and EXT2. Non-allelic homologous recombination (NAHR) mediated by Alu-sequences, microhomology mediated replication dependent recombination (MMRDR) and non-homologous end-joining (NHEJ) were hypothesized as the causal mechanisms in different deletions. Molecular characterization of EXT1- and EXT2-deletion breakpoints in MO-patients indicates that NAHR between Alu-sequences as well as NHEJ are causal and that the majority of these deletions are nonrecurring. These observations emphasize once more the huge genetic variability which is characteristic for MO. To our knowledge, this is the first study characterizing large genomic deletions in EXT1 and EXT2. Show less

Multiple osteochondromas (MO) is a hereditary skeletal disorder characterized by the presence of cartilage capped bony outgrowths at bone surface. Causative mutations in EXT1 or EXT2 genes have been described in 85-90 % of MO cases. However, in about 10-15 % of the MO cases, genomic alterations can not be detected, implying the potential role of other alterations. We have designed a custom-made Agilent oligonucleotide-based microarray, containing 44,000 probes, with tiling coverage of EXT1/2 genes and addition of 68 genes involved in heparan sulfate biosynthesis and other related pathways. Out of the 17 patient samples with previously undetected mutations, a low level of deletion of the EXT1 gene in about 10-15% of the blood cells was detected in two patients and mosaic deletion of the EXT2 was detected in one patient. Here we show that for the first time somatic mosaicism with large genomic deletions as the underlying mechanism in MO formation was identified. We propose that the existence of mosaic mutations and not alterations of other heparan sulfate biosynthesis related genes play a significant role in the development of MO in patients who are tested negative for mutations in Exostosins. Show less

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCalpha in HBMEC. During C. neoformans infection, phosphorylation of PKCalpha was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCalpha isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCalpha colocalized with beta-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCalpha function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCalpha and downstream actin filament activity during the fungal invasion into HBMEC. Show less

Cryptococcus neoformans is a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1 gene, which is required for a capsular layer on the outer cell wall. In this report, we investigate the function of the CPS1 gene and its pathogenesis. We demonstrated that treatment of yeast with either 4-methylumbelliferone or hyaluronidase resulted in a reduction of the level of C. neoformans binding to human brain microvascular endothelial cells (HBMEC). Yeast extracellular structures were also altered accordingly in hyaluronidase-treated cells. Furthermore, observation of yeast strains with different hyaluronic acid contents showed that the ability to bind to HBMEC is proportional to the hyaluronic acid content. A killing assay with Caenorhabditis elegans demonstrated that the CPS1 wild-type strain is more virulent than the cps1Delta strain. When CPS1 is expressed in Saccharomyces cerevisiae and Escherichia coli, hyaluronic acid can be detected in the cells. Additionally, we determined by fluorophore-assisted carbohydrate electrophoretic analysis that hyaluronic acid is a component of the C. neoformans capsule. The size of hyaluronic acid molecules is evaluated by gel filtration and transmission electron microscopy studies. Together, our results support that C. neoformans CPS1 encodes hyaluronic acid synthase and that its product, hyaluronic acid, plays a role as an adhesion molecule during the association of endothelial cells with yeast. Show less

The polysaccharide capsule is known to be the major factor required for the virulence of Cryptococcus neoformans. We have cloned and characterized a gene, designated CPS1, that encodes a protein containing a glycosyltransferase moiety and shares similarity with the type 3 polysaccharide synthase encoded by the cap3B gene of Streptococcus pneumoniae. Cps1p also shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1 gene from a serotype D strain of C. neoformans resulted in a slight reduction of the capsule size as observed by using an India ink preparation. The growth at 37 degrees C was impaired, and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that the conserved glycosyltransferase domains are critical for the ability of the strain to grow at elevated temperatures. A hyaluronan enzyme-linked immunosorbent assay method demonstrated that CPS1 is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild-type and the cps1Delta strains, using three different transmission electron microscopic methods, indicated that the CPS1 gene product is involved in the composition or maintenance of an electron-dense layer between the outer cell wall and the capsule. These and the virulence studies in a mouse model suggested that the CPS1 gene is important in the pathobiology of C. neoformans. Show less

Previous studies with hypertriglyceridemic APOC3 transgenic mice have suggested that apolipoprotein C-III (apoC-III) may inhibit either the apoE-mediated hepatic uptake of TG-rich lipoproteins and/or the lipoprotein lipase (LPL)-mediated hydrolysis of TG. Accordingly, apoC3 knockout (apoC3(-/-)) mice are hypotriglyceridemic. In the present study, we attempted to elucidate the mechanism(s) underlying these phenomena by intercrossing apoC3(-/-) mice with apoE(-/-) mice to study the effects of apoC-III deficiency against a hyperlipidemic background. Similar to apoE(+/+) apoC3(-/-) mice, apoE(-/-)apoC3(-/-) mice exhibited a marked reduction in VLDL cholesterol and TG, indicating that the mechanism(s) by which apoC-III deficiency exerts its lipid-lowering effect act independent of apoE. On both backgrounds, apoC3(-/-) mice showed normal intestinal lipid absorption and hepatic VLDL TG secretion. However, turnover studies showed that TG-labeled emulsion particles were cleared much more rapidly in apoC3(-/-) mice, whereas the clearance of VLDL apoB, as a marker for whole particle uptake by the liver, was not affected. Furthermore, it was shown that cholesteryl oleate-labeled particles were also cleared faster in apoC3(-/-) mice. Thus the mechanisms underlying the hypolipidemia in apoC3(-/-) mice involve both a more efficient hydrolysis of VLDL TG as well as an enhanced selective clearance of VLDL cholesteryl esters from plasma. In summary, our studies of apoC3(-/-) mice support the concept that apoC-III is an effective inhibitor of VLDL TG hydrolysis and reveal a potential regulating role for apoC-III with respect to the selective uptake of cholesteryl esters. Show less

Studies in humans on the in vivo metabolism of apolipoprotein (apo) Cs have been hampered by the highly complex nature of lipoprotein metabolism, which can be influenced by multiple genetic and environmental factors. In order to gain new insights into the function of the individual apoCs in lipoprotein metabolism, several laboratories have created mouse models lacking or overexpressing the respective APOC genes through the technologies of gene targeting and transgenesis. Until now, the only well-established in vivo metabolic function of apoC-I has been its inhibitory action on the uptake of very low-density lipoprotein (VLDL) via hepatic receptors, particularly the low-density lipoprotein (LDL) receptor-related protein. Consequently, the presence of apoC-I on the lipoprotein particle may prolong its residence time in the circulation and subsequently facilitate its conversion to LDL. ApoC-II, on the other hand, is a major activator of lipoprotein lipase, which is required for an efficient processing of triglyceride-rich lipoproteins in the circulation. However, an excess of apoC-II on the lipoprotein particle has been suggested to inhibit the lipoprotein-lipase-mediated hydrolysis of triglycerides. From studies with APOC3 transgenic and ApoC3-knockout mice, it appears that apoC-III inhibits the lipolysis of triglyceride-rich lipoproteins by hampering the interaction of these lipoproteins with the heparan sulfate proteoglycan-lipoprotein lipase complex. Subsequently, the poorly lipolyzed apoC-III-containing lipoprotein particles may accumulate in plasma because of their lower binding affinity towards hepatic receptors due to a change in lipid composition, particle size or the presence of apoC-III on the particle itself. From these data it can thus be concluded that all C apolipoproteins specifically modulate the metabolism of triglyceride-rich lipoproteins, which may contribute to the development of hyperlipidemia and other lipoprotein abnormalities in humans. Show less