Olfr2-positive macrophages originate from monocytes proliferate in situ and present a pro-inflammatory foamy-like phenotype.

AIMS: Olfactory receptor 2 (Olfr2) has been identified in a minimum of 30% of vascular macrophages, and its depletion was shown to reduce atherosclerosis progression. Mononuclear phagocytes, including monocytes and macrophages within the vessel wall, are major players in atherosclerosis. Single-cell RNA sequencing studies revealed that atherosclerotic artery walls encompass several monocytes and vascular macrophages, defining at least nine distinct subsets potentially serving diverse functions in disease progression. This study investigates the functional phenotype and ontogeny of Olfr2-expressing vascular macrophages in atherosclerosis. METHODS AND RESULTS: Olfr2+ macrophages rapidly increase in Apoe-/- mice's aorta when fed a Western diet (WD). Mass cytometry showed that Olfr2+ cells are clustered within the CD64 high population and enriched for CD11c and Ccr2 markers. Olfr2+ macrophages express many pro-inflammatory cytokines, including Il1b, Il6, Il12, and Il23, and chemokines, including Ccl5, Cx3cl1, Cxcl9, and Ccl22. By extracting differentially expressed genes from bulk RNA sequencing (RNA-seq) of Olfr2+ vs. Olfr2- macrophages, we defined a signature that significantly mapped to single-cell data of plaque myeloid cells, including monocytes, subendothelial MacAir, and Trem2Gpnmb foamy macrophages. By adoptive transfer experiments, we identified that Olfr2 competent monocytes from CD45.1Apoe-/-Olfr2+/+ mice transferred into CD45.2Apoe-/-Olfr2-/- recipient mice fed WD for 12 weeks, accumulate in the atherosclerotic aorta wall already at 72 h, and differentiate in macrophages. Olfr2+ macrophages showed significantly increased BrdU incorporation compared to Olfr2- macrophages. Flow cytometry confirmed that at least 50% of aortic Olfr2+ macrophages are positive for BODIPY staining and have increased expression of both tumour necrosis factor and interleukin 6 compared to Olfr2- macrophages. Gene set enrichment analysis of the Olfr2+ macrophage signature revealed a similar enrichment pattern in human atherosclerotic plaques, particularly within foamy/TREM2hi-Mφ and monocytes. CONCLUSIONS: In summary, we conclude that Olfr2+ macrophages in the aorta originate from monocytes and can accumulate at the early stages of disease progression. These cells can undergo differentiation into MacAir and Trem2Gpnmb foamy macrophages, exhibiting proliferative and pro-inflammatory potentials. This dynamic behaviour positions them as key influencers in shaping the myeloid landscape within the atherosclerotic plaque.

Identification of human exTreg cells as CD16+CD56+ cytotoxic CD4+ T cells.

In atherosclerosis, some regulatory T (Treg) cells become exTreg cells. We crossed inducible Treg and exTreg cell lineage-tracker mice (FoxP3eGFP-Cre-ERT2ROSA26CAG-fl-stop-fl-tdTomato) to atherosclerosis-prone Apoe-/- mice, sorted Treg cells and exTreg cells and determined their transcriptomes by bulk RNA sequencing (RNA-seq). Genes that were differentially expressed between mouse Treg cells and exTreg cells and filtered for their presence in a human single-cell RNA-sequencing (scRNA-seq) panel identified exTreg cell signature genes as CST7, NKG7, GZMA, PRF1, TBX21 and CCL4. Projecting these genes onto the human scRNA-seq with CITE-seq data identified human exTreg cells as CD3+CD4+CD16+CD56+, which was validated by flow cytometry. Bulk RNA-seq of sorted human exTreg cells identified them as inflammatory and cytotoxic CD4+T cells that were significantly distinct from both natural killer and Treg cells. DNA sequencing for T cell receptor-ß showed clonal expansion of Treg cell CDR3 sequences in exTreg cells. Cytotoxicity was functionally demonstrated in cell killing and CD107a degranulation assays, which identifies human exTreg cells as cytotoxic CD4+T cells.

A humanized ß2 integrin knockin mouse reveals localized intra- and extravascular neutrophil integrin activation in vivo.

ß2 integrins are leukocyte-specific adhesion molecules that are essential for leukocyte recruitment. The lack of tools for reporting ß2 integrin activation in mice hindered the study of ß2 integrin-related immune responses in vivo. Here, we generated a humanized ß2 integrin knockin mouse strain by targeting the human ß2 integrin coding sequence into the mouse Itgb2 locus to enable imaging of ß2 integrin activation using the KIM127 (extension) and mAb24 (high-affinity) reporter antibodies. Using a CXCL1-induced acute inflammation model, we show the local dynamics of ß2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins are highly concentrated in a small area at the rear of arresting neutrophils in vivo. In a high-dose lipopolysaccharide model, we find that ß2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice enable studies of ß2 integrin activation in vivo.

Antibacterial Mechanism of Cinnamaldehyde: Modulation of Biosynthesis of Phosphatidylethanolamine and Phosphatidylglycerol in Staphylococcus aureus and Escherichia coli.

Cinnamaldehyde is a natural antimicrobial food preservative. Previous studies have suggested that cinnamaldehyde interacts with the cell membrane, but the molecular targets of cinnamaldehyde action on foodborne pathogens are still unclear. In this study, the structural changes of Staphylococcus aureus and Escherichia coli cells were observed after cinnamaldehyde treatment. Then, quantitative real-time polymerase chain reaction (PCR) and parallel reaction monitoring were used for determining the effects of cinnamaldehyde treatment of these bacteria on the expression of genes and proteins associated with glycerophospholipid biosynthesis. Changes in fatty acids (raw materials for the biosynthesis of glycerophospholipids) and glycerophospholipids in S. aureus and E. coli after cinnamaldehyde treatment were analyzed to confirm the results of gene and protein expression experiments. Cinnamaldehyde regulated the glycerophospholipid biosynthesis pathways of these foodborne pathogens, mainly targeting phosphatidylglycerol and phosphatidylethanolamine, which resulted in the disruption of cell membrane integrity.

Carotenoid biosynthesis and the evolution of carotenogenesis genes in rust fungi.

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl-geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the "scabrum" rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.

Novel Antioxidant Peptides Purified from Mulberry (Morus atropurpurea Roxb.) Leaf Protein Hydrolysates with Hemolysis Inhibition Ability and Cellular Antioxidant Activity.

Neutrase-hydrolysates hydrolyzed from mulberry leaf proteins were separated by ion exchange chromatography, gel filtration chromatography, and semipreparative reverse-phase HPLC. Purified fractions were analyzed for their radical scavenging activity, hemolysis inhibition ability, and cellular antioxidant activity (CAA). Three new antioxidant peptides, P1 (SVL, 317 Da), P2 (EAVQ, 445 Da), and P3 (RDY, 452 Da), were obtained from the most active HPLC fraction (R1) and identified using UPLC-QTOF-MS. These three peptides were then synthesized, and their antioxidant activities were analyzed. P1 and P2 had no ability to inhibit hemolysis of erythrocytes but did show antioxidant activity on HepG2 cells. P3 showed the highest hemolysis inhibition ability (92%) and CAA value (2204 µM QE/100 g peptide). The Tyr residues at the C-terminal region play an important role in the antioxidant activity in P3. Thus, the natural peptide R1 and synthesized P3 could be used as antioxidants and might be promising components of functional foods.

Carotenoid complement of rust spores: Variation among species and pathotype.

Rust fungi, which are responsible for some of the world's most destructive plant diseases, are named for the distinctive rusty colour of one or more of their spore forms. Rust spore pigments are thought to provide protection against UV radiation and oxidative stress, and may act as virulence factors. However, with the exception of daisy rust spores, the identity and relative abundance of the carotenoids in the rust spore cytoplasm have not been investigated using modern analytical methods, and little is known about the dependence of the carotenoid complement on species, pathotype, spore-colour mutations and season. We developed and validated a method to separate, identify and quantify rust carotenoids by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with mass spectrometry. The carotenoids identified were lycopene, γ-carotene, ß-carotene and phytoene. Rates of carotenoid degradation depended greatly on spore storage conditions, with freezing at -80 °C providing optimal stability. Carotenoid profiles of 103 isolates from 14 rust species were compared, showing that the ratio γ-carotene:ß-carotene varied substantially among species. Total carotenoid content was generally lower in spring than in autumn (Sydney, Australia)-possibly due to differences in solar exposure-but the percentage of individual carotenoids was relatively stable. Among the colour mutants tested, chocolate mutants of Puccinia graminis f. sp. tritici (wheat stem rust) contained no carotenoid pigments, while albino mutants of P. striiformis f. sp. tritici (wheat stripe rust) contained only phytoene, a colourless carotenoid. We discuss our results in terms of the biogenesis and biological functions of carotenoids in rust fungi.

Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives' Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps.

This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively.

Characterisation and oxidation stability of monoacylglycerols from partially hydrogenated corn oil.

This study reported the characterisation of some types of monoacylglycerols (MAGs) obtained by the glycerolysis of different partially hydrogenated corn oils (PHCOs) catalysed by Al2O3 loaded with K2CO3 (K2CO3/Al2O3) under the previous selected conditions. A two-stage molecular distillation method of purifying the MAGs was introduced, and the obtained MAG products were more than 90.0 wt.% pure. The fatty acid composition of corn oil significantly changed after hydrogenation sequentially catalysed by Pricat™ Ni catalysts (9908 Ni/kieselguhr and 9920 Ni/Al2O3). The PHCO samples generated typical structures with ß'-form crystals. Moreover, the melting regions of all hydrogenated samples and their MAGs shifted to higher temperatures. The oxidation stability of MAGs has been significantly increased using hydrogenation to change the fatty acid composition.