Menopause and facial skin microbiomes: a pilot study revealing novel insights into their relationship.

Introduction: The human skin microbial composition is affected by age. Previous studies reported skin microbiome diversity shifts between elderly and significantly younger subjects. Some studies implied that menopausal status, which is inherently linked to age, could be associated with changes in skin microbial compositions. Nevertheless, the influence of menopausal status on skin microbiome profiles while minimizing the impact of aging-associated changes in skin parameters still needs further clarification. Methods: We performed an observational study on healthy Caucasian female volunteers, which were grouped according to their pre- or postmenopausal status. Bacterial community structures on facial skin were analyzed using 16S rRNA gene sequencing. Cutometer® measurements were performed to evaluate aging-associated changes in facial skin biophysical properties. Results: The relative abundance of the lipophilic Cutibacterium genus was decreased, and bacterial diversity was increased in skin samples of postmenopausal volunteers. The mean age difference between examined groups in this study was 12.4 years only. Accordingly, Cutometer® measurements revealed no differences in aging-associated skin biophysical parameters between pre- and postmenopausal groups. Consequently, no correlation was detected between Shannon diversity and measured age-dependent biomechanical properties of facial skin. Discussion: These findings are in line with previous studies, which investigated the wide-ranging impact of chronological aging on skin microbial communities. However, this work reports for the first time a direct association between menopausal status and facial microbiomes on skin of similarly aged study participants, and hence uncouples aging-associated skin biophysical parameters, such as viscoelastic properties, from the equation. These findings open avenues for the development of microbiome-targeting strategies for treatment of menopause-associated skin disorders.

Host/Malassezia Interaction: A Quantitative, Non-Invasive Method Profiling Oxylipin Production Associates Human Skin Eicosanoids with Malassezia.

Malassezia are common components of human skin, and as the dominant human skin eukaryotic microbe, they take part in complex microbe-host interactions. Other phylogenetically related fungi (including within Ustilagomycotina) communicate with their plant host through bioactive oxygenated polyunsaturated fatty acids, generally known as oxylipins, by regulating the plant immune system to increase their virulence. Oxylipins are similar in structure and function to human eicosanoids, which modulate the human immune system. This study reports the development of a highly sensitive mass-spectrometry-based method to capture and quantify bioactive oxygenated polyunsaturated fatty acids from the human skin surface and in vitro Malassezia cultures. It confirms that Malassezia are capable of synthesizing eicosanoid-like lipid mediators in vitro in a species dependent manner, many of which are found on human skin. This method enables sensitive identification and quantification of bioactive lipid mediators from human skin that may be derived from metabolic pathways shared between skin and its microbial residents. This enables better cross-disciplinary and detailed studies to dissect the interaction between Malassezia and human skin, and to identify potential intervention points to promote or abrogate inflammation and to improve human skin health.

Fungal Jasmonate as a Novel Morphogenetic Signal for Pathogenesis.

A key question that has remained unanswered is how pathogenic fungi switch from vegetative growth to infection-related morphogenesis during a disease cycle. Here, we identify a fungal oxylipin analogous to the phytohormone jasmonic acid (JA), as the principal regulator of such a developmental switch to isotropic growth and pathogenicity in the rice-blast fungus Magnaporthe oryzae. Using specific inhibitors and mutant analyses, we determined the molecular function of intrinsic jasmonates during M. oryzae pathogenesis. Loss of 12-Oxo-phytodienoic Acid (OPDA) Reductase and/or consequent reduction of jasmonate biosynthesis, prolonged germ tube growth and caused delayed initiation and improper development of infection structures in M. oryzae, reminiscent of phenotypic defects upon impaired cyclic AMP (cAMP) signaling. Chemical- or genetic-complementation completely restored proper vegetative growth and appressoria in opr1Δ. Mass spectrometry-based quantification revealed increased OPDA accumulation and significantly decreased jasmonate levels in opr1Δ. Most interestingly, exogenous jasmonate restored proper appressorium formation in pth11Δ that lacks G protein/cAMP signaling; but failed to do so in the Mitogen-activated protein (MAP) kinase mutants. Epistasis analysis placed jasmonate upstream of the cAMP pathway in rice blast. Mechanistically, intrinsic jasmonate orchestrates timely cessation of the vegetative phase and induces pathogenic development via a complex regulatory interaction with the cAMP-PKA cascade and redox signaling in rice blast.

Seipin negatively regulates sphingolipid production at the ER-LD contact site.

Seipin is known for its critical role in controlling lipid droplet (LD) assembly at the LD-forming subdomain of the endoplasmic reticulum (ER). Here, we identified a new function of seipin as a negative regulator for sphingolipid production. We show that yeast cells lacking seipin displayed altered sensitivity to sphingolipid inhibitors, accumulated sphingoid precursors and intermediates, and increased serine palmitoyltransferase (SPT) and fatty acid (FA) elongase activities. Seipin associated with SPT and FA elongase, and the interaction was reduced by inhibitors for sphingolipid synthesis in a concentration-dependent manner. We further show that the interactions of seipin with SPT and FA elongase occurred at ER-LD contacts and were likely regulated differentially. Further evidence indicated that LD biogenesis was intact when SPT activity was blocked, whereas excess sphingoid intermediates may affect LD morphology. Expression of human seipin rescued the altered sphingolipids in yeast seipin mutants, suggesting that the negative regulation of sphingolipid synthesis by seipin is likely an evolutionarily conserved process.

SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase.

Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. Here, we identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Overexpression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin-/- mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients.

Topology of the microsomal glycerol-3-phosphate acyltransferase Gpt2p/Gat1p of Saccharomyces cerevisiae.

All glycerophospholipids are made from phosphatidic acid, which, according to the traditional view, is generated at the cytosolic surface of the ER. In yeast, phosphatidic acid is synthesized de novo by two acyl-CoA-dependent acylation reactions. The first is catalysed by one of the two homologous glycerol-3-phosphate acyltransferases Gpt2p/Gat1p and Sct1p/Gat2p, the second by one of the two 1-acyl-sn-glycerol-3-phosphate acyltransferases Slc1p and Ale1p/Slc4p. To study the biogenesis and topology of Gpt2p we observed the location of dual topology reporters inserted after various transmembrane helices. Moreover, using microsomes, we probed the accessibility of natural and substituted cysteine residues to a membrane impermeant alkylating agent and tested the protease sensitivity of various epitope tags inserted into Gpt2p. Finally, we assayed the sensitivity of the acyltransferase activity to membrane impermeant agents targeting lysine residues. By all these criteria we find that the most conserved motifs of Gpt2p and its functionally relevant lysines are oriented towards the ER lumen. Thus, the first step in biosynthesis of phosphatidic acid in yeast seems to occur in the ER lumen and substrates may have to cross the ER membrane.

Topology of 1-acyl-sn-glycerol-3-phosphate acyltransferases SLC1 and ALE1 and related membrane-bound O-acyltransferases (MBOATs) of Saccharomyces cerevisiae.

In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed.

On-line flow-through extraction-preconcentration-large volume injection-RP LC for trace determination of pyrethroids in Slovak soil.

A rapid micro-analytical multiresidue method was developed for analysis of pyrethroids (kadethrin K, cypermethrin C and permethrin P) in soil micro-sample (200 mg). It uses on-line flow-through extraction of soil micro-samples (packed into a short glass column) with a methanol-aqueous citric acid buffer mixture, successive on-line SPE preconcentration of analytes from the extract and on-line RP-HPLC analysis with UV photometric detection. The separation of pyrethroids is performed on a Purospher RP-18e column with methanol/water as mobile phase. Effects of sorbent placed at the bottom of a short column holding the soil sample and different kinds of on-line SPE columns were tested. Besides, the influence of volume of the effluent on the pyrethroids recovery was also studied. Calibration curves were linear over the range assayed from 0.01 to 0.2 microg/mL with correlation coefficients of linear regression (least-squares method) in the range 0.998-0.999. Recovery studies were carried out at 0.25-1.00 microg/g dry soil fortification level and obtained recoveries were for K 81-84%, C 56-59% and for P 58-63%. Achieved LOD (confidence band) of studied pyrethroids were for large-volume injection (1 mL) 4.5 ng K, 3.7 ng C, 3.6 ng P or 27 ng/g K, 32 ng/g C and 29 ng/g P in dry soil "solid sampling HPLC".

The Gup1 homologue of Trypanosoma brucei is a GPI glycosylphosphatidylinositol remodelase.

Glycosylphosphatidylinositol (GPI) lipids of Trypanosoma brucei undergo lipid remodelling, whereby longer fatty acids on the glycerol are replaced by myristate (C14:0). A similar process occurs on GPI proteins of Saccharomyces cerevisiae where Per1p first deacylates, Gup1p subsequently reacylates the anchor lipid, thus replacing a shorter fatty acid by C26:0. Heterologous expression of the GUP1 homologue of T. brucei in gup1Delta yeast cells partially normalizes the gup1Delta phenotype and restores the transfer of labelled fatty acids from Coenzyme A to lyso-GPI proteins in a newly developed microsomal assay. In this assay, the Gup1p from T. brucei (tbGup1p) strongly prefers C14:0 and C12:0 over C16:0 and C18:0, whereas yeast Gup1p strongly prefers C16:0 and C18:0. This acyl specificity of tbGup1p closely matches the reported specificity of the reacylation of free lyso-GPI lipids in microsomes of T. brucei. Depletion of tbGup1p in trypanosomes by RNAi drastically reduces the rate of myristate incorporation into the sn-2 position of lyso-GPI lipids. Thus, tbGup1p is involved in the addition of myristate to sn-2 during GPI remodelling in T. brucei and can account for the fatty acid specificity of this process. tbGup1p can act on GPI proteins as well as on GPI lipids.

Structure-function analysis of the endoplasmic reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain.

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b'). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb', we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb', the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.