Sterol Biosynthesis in Four Green Algae: A Bioinformatic Analysis of the Ergosterol Versus Phytosterol Decision Point.

Animals and fungi produce cholesterol and ergosterol, respectively, while plants produce the phytosterols stigmasterol, campesterol, and ß-sitosterol in various combinations. The recent sequencing of many algal genomes allows the detailed reconstruction of the sterol metabolic pathways. Here, we characterized sterol synthesis in two sequenced Chlorella spp., the free-living C. sorokiniana, and symbiotic C. variabilis NC64A. Chlamydomonas reinhardtii was included as an internal control and Coccomyxa subellipsoidea as a plant-like outlier. We found that ergosterol was the major sterol produced by Chlorella spp. and C. reinhardtii, while C. subellipsoidea produced the three phytosterols found in plants. In silico analysis of the C. variabilis NC64A, C. sorokiniana, and C. subellipsoidea genomes identified 22 homologs of sterol biosynthetic genes from Arabidopsis thaliana, Saccharomyces cerevisiae, and C. reinhardtii. The presence of CAS1, CPI1, and HYD1 in the four algal genomes suggests the higher plant cycloartenol branch for sterol biosynthesis, confirming that algae and fungi use different pathways for ergosterol synthesis. Phylogenetic analysis for 40 oxidosqualene cyclases (OSCs) showed that the nine algal OSCs clustered with the cycloartenol cyclases, rather than the lanosterol cyclases, with the OSC for C. subellipsoidea positioned in between the higher plants and the eight other algae. With regard to why C. subellipsoidea produced phytosterols instead of ergosterol, we identified 22 differentially conserved positions where C. subellipsoidea CAS and A. thaliana CAS1 have one amino acid while the three ergosterol producing algae have another. Together, these results emphasize the position of the unicellular algae as an evolutionary transition point for sterols.

Maternal HIV status skews transcriptomic response in infant cord blood monocytes exposed to Bacillus Calmette--Guerín.

OBJECTIVES: HIV-exposed uninfected (HEU) infants exhibit altered vaccine responses and an increased mortality compared with HIV-unexposed infants. Here, vaccine responses in HEU and HIV-unexposed cord blood monocytes (CBMs) were assessed following Bacillus Calmette--Guerín (BCG) treatment. DESIGN: Innate responses to in-vitro BCG treatment were assessed through transcriptional profiling using CBMs obtained from a Nigerian cohort of HIV-infected and uninfected women. METHODS: HIV-unexposed (n = 9) and HEU (n = 10) infant CBMs were treated with BCG and transcriptionally profiled with the Nanostring nCounter platform. Differential expression and pathway enrichment analyses were performed, and transcripts were identified with enhanced or dampened BCG responses. RESULTS: Following BCG stimulation, several pathways associated with inflammatory gene expression were upregulated irrespective of HIV exposure status. Both HIV-unexposed and HEU monocytes increased expression of several cytokines characteristic of innate BCG responses, including IL1ß, TNFα, and IL-6. Using differential expression analysis, we identified genes significantly upregulated in HEU compared with HIV-unexposed monocytes including monocyte chemokine CCL7 and anti-inflammatory cytokine TNFAIP6. In contrast, genes significantly upregulated in HIV-unexposed compared with HEU monocytes include chemokine CCL3 and cytokine IL23A, both of which influence anti-mycobacterial T-cell responses. Finally, two genes, which regulate prostaglandin production, CSF2 and PTGS2, were also more significantly upregulated in the HIV-unexposed cord blood indicating that inflammatory mediators are suppressed in the HEU infants. CONCLUSION: HEU monocytes exhibit altered induction of several key innate immune responses, providing mechanistic insights into dysregulated innate response pathways that can be therapeutically targeted to improve vaccine responses in HEU infants.

Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host-virus interactions.

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins.