Profiling of epidermal lipids in a mouse model of dermatitis: Identification of potential biomarkers.

Lipids are important structural and functional components of the skin. Alterations in the lipid composition of the epidermis are associated with inflammation and can affect the barrier function of the skin. SHARPIN-deficient cpdm mice develop a chronic dermatitis with similarities to atopic dermatitis in humans. Here, we used a recently-developed approach named multiple reaction monitoring (MRM)-profiling and single ion monitoring to rapidly identify discriminative lipid ions. Shorter fatty acyl residues and increased relative amounts of sphingosine ceramides were observed in cpdm epidermis compared to wild type mice. These changes were accompanied by downregulation of the Fasn gene which encodes fatty acid synthase. A profile of diverse lipids was generated by fast screening of over 300 transitions (ion pairs). Tentative attribution of the most significant transitions was confirmed by product ion scan (MS/MS), and the MRM-profiling linear intensity response was validated with a C17-ceramide lipid standard. Relative quantification of sphingosine ceramides CerAS(d18:1/24:0)2OH, CerAS(d18:1/16:0)2OH and CerNS(d18:1/16:0) discriminated between the two groups with 100% accuracy, while the free fatty acids cerotic acid, 16-hydroxy palmitic acid, and docosahexaenoic acid (DHA) had 96.4% of accuracy. Validation by liquid chromatography tandem mass spectrometry (LC-MS/MS) of the above-mentioned ceramides was in agreement with MRM-profiling results. Identification and rapid monitoring of these lipids represent a tool to assess therapeutic outcomes in SHARPIN-deficient mice and other mouse models of dermatitis and may have diagnostic utility in atopic dermatitis. PLoS One 2018 Apr 26; 13(4):e0196595

Multiple Reaction Monitoring Profiling (MRM-Profiling) of Lipids To Distinguish Strain-Level Differences in Microbial Resistance in Escherichia coli

The worldwide increase in antimicrobial resistance is due to antibiotic overuse in agriculture and overprescription in medicine. For appropriate and timely patient support, faster diagnosis of antimicrobial resistance is required. Current methods for bacterial identification rely on genomics and proteomics and use comparisons with databases of known strains, but the diagnostic value of metabolites and lipids has not been explored significantly. Standard mass spectrometry/chromatography methods involve multiple dilutions during sample preparation and separation. To increase the amount of chemical information acquired and the speed of analysis of lipids, multiple reaction monitoring profiling (MRM-Profiling) has been applied. The MRM-Profiling workflow includes a discovery stage and a screening stage. The discovery stage employs precursor (PREC) ion and neutral loss (NL) scans to screen representative pooled samples for functional groups associated with particular lipid classes. The information from the first stage is organized in precursor/product ion pairs, or MRMs, and the screening stage rapidly interrogates individual samples for these MRMs. In this study, we performed MRM-Profiling of lipid extracts from four different strains of Escherichia coli cultured with amoxicillin or amoxicillin/clavulanate, a β-lactam and β-lactamase inhibitor, respectively. t tests, analysis of variance and receiver operating characteristic (ROC) curves were used to determine the significance of each MRM. Principal component analysis was applied to distinguish different strains cultured under conditions that allowed or disallowed development of bacterial resistance. The results demonstrate that MRM-Profiling distinguishes the lipid profiles of resistant and nonresistant E. coli strains

Global proteomics insights for a novel small compound targeting the non-integrin Laminin Receptor in a macrophage cell model

Introduction: Monocytes and macrophages are the first barrier of the innate immune system, which interact with agents causing osteoarthritis or other conditions, leading to the release of proinflammatory mediators that exacerbate inflammation.Methods: The aim of this study was to investigate the proteomic changes in THP-1 monocytes differentiated to macrophages, pre- or -post small compound treatments and in the presence or absence of a proinflammatory stimulus, Lipopolysaccharide (LPS). This study aimed to discover and isolate small compounds that mimic the interaction between Pigment derived growth factor (PEDF) and its 37/67 kDa Laminin receptor (LR) with potential anti-inflammatory activity.Results: Our results suggested that novel compounds targeting the LR-PEDF interface can be useful for modulating anti-inflammatory effects. Several compounds were selected based on in silico docking at the PEDF/LR interface and examined for their ability to reduce IL-1β expression in a macrophage cell model. Compound C3 showed the highest efficacy in reducing IL-1β expression in the presence of LPS proinflammatory stimulus. Proteomics analysis revealed that C3 treatment altered the global proteomic profile of THP-1 activated macrophages, affecting pathways such as MYC targets, oxidative phosphorylation, and mTORC1 signaling.Discussion: The analysis also highlighted the involvement of key regulators, including RPSA and MYC, and their interactions with other proteins such as ribosome proteins and cell cycle regulators. Furthermore, the downregulated proteome analysis revealed shared and unique pathways affected by the treatments, including processes related to actin cytoskeleton, translation, and the inflammatory response. Protein-protein interaction networks suggested the potential involvement of transcription factors like MYC and the interconnectedness of signaling pathways in mediating such as the effects of the treatments. Overall, these findings provide valuable insights into the potential anti-inflammatory activity and underlying mechanisms of compound C3, emphasizing its relevance for further investigation in the context of inflammatory conditions

Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoiddependent axial patterning in metazoans

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

Abstract\ud \ud \ud \ud Background\ud \ud Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus.\ud \ud \ud \ud Methods\ud \ud One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay.\ud \ud \ud \ud Results\ud \ud We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls.\ud \ud \ud \ud Conclusion\ud \ud In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/000090; 00/030722; 01/142381; 02/113402; 03/099982; 04/116068; 04/157044 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/20078. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/00009-0; 00/03072-2; 01/14238-1; 02/11340-2; 03/09998-2; 04/11606-8; 04/15704-4 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/2007-8. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process

Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicleassociated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasm

Histoplasma capsulatum Heat-Shock 60 Orchestrates the Adaptation of the Fungus to Temperature Stress

Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins. Hsps are essential regulators of diverse constitutive metabolic processes and are markedly upregulated during stress. A 62 kDa Hsp (Hsp60) of Histoplasma capsulatum (Hc) is an immunodominant antigen and the major surface ligand to CR3 receptors on macrophages. However little is known about the function of this protein within the fungus. We characterized Hc Hsp60-protein interactions under different temperature to gain insights of its additional functions oncell wall dynamism, heat stress and pathogenesis. We conducted co-immunoprecipitations with antibodies to Hc Hsp60 using cytoplasmic and cell wall extracts. Interacting proteins were identified by shotgun proteomics. For the cell wall, 84 common interactions were identified among the 3 growth conditions, including proteins involved in heat-shock response, sugar and amino acid/protein metabolism and cell signaling. Unique interactions were found at each temperature [30°C (81 proteins), 37°C (14) and 37/40°C (47)]. There were fewer unique interactions in cytoplasm [30°C (6), 37°C (25) and 37/40°C (39)] and four common interactions, including additional Hsps and other known virulence factors. These results show the complexity of Hsp60 function and provide insights into Hc biology, which may lead to new avenues for the management of histoplasmosis

Sequence-specific reconstruction from fragmentary databases using seed sequences: implementation and validation on SAGE, proteome and generic sequencing data

Motivation: DNA assembly programs classically perform an all-against-all comparison of reads to identify overlaps, followed by a multiple sequence alignment and generation of a consensus sequence. If the aim is to assemble a particular segment, instead of a whole genome or transcriptome, a target-specific assembly is a more sensible approach. GenSeed is a Perl program that implements a seed-driven recursive assembly consisting of cycles comprising a similarity search, read selection and assembly. The iterative process results in a progressive extension of the original seed sequence. GenSeed was tested and validated on many applications, including the reconstruction of nuclear genes or segments, full-length transcripts, and extrachromosomal genomes. The robustness of the method was confirmed through the use of a variety of DNA and protein seeds, including short sequences derived from SAGE and proteome projects