Activation of ALOX12 by a multi-organelle-orienting photosensitizer drives ACSL4-independent cell ferroptosis.

Ferroptosis is a recently-defined tumor suppression mechanism, but the sensitivity of many tumorigenic cells to ferroptosis is limited by their deficient expression of acyl-CoA synthetase long-chain family member 4 (ACSL4). Here, we report the discovery of a photosensitizer, namely TPCI, which can evoke ACSL4-independent ferroptosis of cancer cells in photodynamic therapy. Through co-localization with 12-lipoxygenase (ALOX12) in multiple subcellular organelles, TPCI activates ALOX12 to generate lipid reactive oxygen species in large quantity and trigger cell ferroptosis. Intriguingly, confining TPCI exclusively in lysosomes switches the cell death from ferroptosis to apoptosis. More strikingly, the ferroptosis mediated by TPCI-induced ALOX12 activation does not require the participation of ACSL4. Therefore, our study identifies TPCI as the first ALOX12 activator to induce ferroptosis independent of ACSL4, which renders a viable therapeutic approach on the basis of distinct ferroptosis of cancer cells, regardless their ACSL4 expressions.

The Human Milk Oligosaccharides 3-FL, Lacto-N-Neotetraose, and LDFT Attenuate Tumor Necrosis Factor-α Induced Inflammation in Fetal Intestinal Epithelial Cells In Vitro through Shedding or Interacting with Tumor Necrosis Factor Receptor 1.

SCOPE: Human milk oligosaccharides (hMOs) can attenuate inflammation by modulating intestinal epithelial cells, but the mechanisms of action are not well-understood. Here, the effects of hMOs on tumor necrosis factor-α (TNF-α) induced inflammatory events in gut epithelial cells are studied. METHODS AND RESULTS: The modulatory effects of 2'-fucosyllactose, 3-fucosyllactose (3-FL), 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), lactodifucotetraose (LDFT), and lacto-N-triaose (LNT2) on immature (FHs 74 Int) and adult (T84) intestinal epithelial cells with or without TNF-α are determined. Interleukin-8 (IL-8) secretion in FHs 74 Int and T84 are quantified to determine hMO induced attenuation of inflammatory events by ELISA. 3-FL, LNnT, and LDFT significantly attenuate TNF-α induced inflammation in FHs 74 Int, while LNT2 induces IL-8 secretion in T84. In addition, microscale thermophoresis assays and ELISA are used to study the possible mechanisms of interaction between effective hMOs and tumor necrosis factor receptor 1 (TNFR1). 3-FL, LNnT, and LDFT exert TNFR1 ectodomain shedding while LNnT also shows binding affinity to TNFR1 with a Kd of 900 ± 660 nM. CONCLUSION: The findings indicate that specific hMO types attenuate TNF-α induced inflammation in fetal gut epithelial cells through TNFR1 in a hMO structure-dependent fashion suggest possibilities to apply hMOs in management of TNF-α dependent diseases.

More than sugar in the milk: human milk oligosaccharides as essential bioactive molecules in breast milk and current insight in beneficial effects.

Human milk is the gold standard for newborn infants. Breast milk not only provides nutrients, it also contains bioactive components that guide the development of the infant's intestinal immune system, which can have a lifelong effect. The bioactive molecules in breast milk regulate microbiota development, immune maturation and gut barrier function. Human milk oligosaccharides (hMOs) are the most abundant bioactive molecules in human milk and have multiple beneficial functions such as support of growth of beneficial bacteria, anti-pathogenic effects, immune modulating effects, and stimulation of intestine barrier functions. Here we critically review the current insight into the benefits of bioactive molecules in mother milk that contribute to neonatal development and focus on current knowledge of hMO-functions on microbiota and the gastrointestinal immune barrier. hMOs produced via genetically engineered microorganisms are now applied in infant formulas to mimic the nutritional composition of breast milk as closely as possible, and their prospects and scientific challenges are discussed in depth.

Effects of Different Human Milk Oligosaccharides on Growth of Bifidobacteria in Monoculture and Co-culture With Faecalibacterium prausnitzii.

Human milk oligosaccharides (hMOs) are important bioactive components in mother's milk contributing to infant health by supporting colonization and growth of gut microbes. In particular, Bifidobacterium genus is considered to be supported by hMOs. Approximately 200 different hMOs have been discovered and characterized, but only a few abundant hMOs can be produced in sufficient amounts to be applied in infant formula. These hMOs are usually supplied in infant formula as single molecule, and it is unknown which and how individual hMOs support growth of individual gut bacteria. To investigate how individual hMOs influence growth of several relevant intestinal bacteria species, we studied the effects of three hMOs (2'-fucosyllactose, 3-fucosyllactose, and 6'-sialyllactose) and an hMO acid hydrolysate (lacto-N-triose) on three Bifidobacteria and one Faecalibacterium and introduced a co-culture system of two bacterial strains to study possible cross-feeding in presence and absence of hMOs. We observed that in monoculture, Bifidobacterium longum subsp. infantis could grow well on all hMOs but in a structure-dependent way. Faecalibacterium prausnitzii reached a lower cell density on the hMOs in stationary phase compared to glucose, while B. longum subsp. longum and Bifidobacterium adolescentis were not able to grow on the tested hMOs. In a co-culture of B. longum subsp. infantis with F. prausnitzii, different effects were observed with the different hMOs; 6'-sialyllactose, rather than 2'-fucosyllactose, 3-fucosyllactose, and lacto-N-triose, was able to promote the growth of B. longum subsp. infantis. Our observations demonstrate that effects of hMOs on the tested gut microbiota are hMO-specific and provide new means to support growth of these specific beneficial microorganisms in the intestine.

Human Milk Oligosaccharides Mediate the Crosstalk Between Intestinal Epithelial Caco-2 Cells and Lactobacillus PlantarumWCFS1in an In Vitro Model with Intestinal Peristaltic Shear Force.

BACKGROUND: The intestinal epithelial cells, food molecules, and gut microbiota are continuously exposed to intestinal peristaltic shear force. Shear force may impact the crosstalk of human milk oligosaccharides (hMOs) with commensal bacteria and intestinal epithelial cells. OBJECTIVES: We investigated how hMOs combined with intestinal peristaltic shear force impact intestinal epithelial cells and crosstalk with a commensal bacterium. METHODS: We applied the Ibidi system to mimic intestinal peristaltic shear force. Caco-2 cells were exposed to a shear force (5 dynes/cm2) for 3 d, and then stimulated with the hMOs, 2'-fucosyllactose (2'-FL), 3-FL, and lacto-N-triose II (LNT2). In separate experiments, Lactobacillus plantarumWCFS1 adhesion to Caco-2 cells was studied with the same hMOs and shear force. Effects were tested on gene expression of glycocalyx-related molecules (glypican 1 [GPC1], hyaluronan synthase 1 [HAS1], HAS2, HAS3, exostosin glycosyltransferase 1 [EXT1], EXT2), defensin ß-1 (DEFB1), and tight junction (tight junction protein 1 [TJP1], claudin 3 [CLDN3]) in Caco-2 cells. Protein expression of tight junctions was also quantified. RESULTS: Shear force dramatically decreased gene expression of the main enzymes for making glycosaminoglycan side chains (HAS3 by 43.3% and EXT1 by 68.7%) (P <0.01), but did not affect GPC1 which is the gene responsible for the synthesis of glypican 1 which is a major protein backbone of glycocalyx. Expression of DEFB1, TJP1, and CLDN3 genes was decreased 60.0-94.9% by shear force (P <0.001). The presence of L. plantarumWCFS1 increased GPC1, HAS2, HAS3, and ZO-1 expression by 1.78- to 3.34-fold (P <0.05). Under shear force, all hMOs significantly stimulated DEFB1 and ZO-1, whereas only 3-FL and LNT2 enhanced L. plantarumWCFS1 adhesion by 1.85- to 1.90-fold (P <0.01). CONCLUSIONS: 3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarumWCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs.

Human Milk Oligosaccharides Differently Modulate Goblet Cells Under Homeostatic, Proinflammatory Conditions and ER Stress.

SCOPE: Human milk oligosaccharides (hMOs) have beneficial effects on intestinal barrier function, but the mechanisms of action are not well understood. Here, the effects of hMOs on goblet cells, which indicate that some hMOs may enhance mucus barrier function through direct modulation of goblet cell function, are studied. METHODS AND RESULTS: The modulatory effects of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-triaose II (LNT2), and galacto-oligosaccharides (GOS) on the expression of goblet cell secretory related genes MUC2, TFF3, and RETNLB, and the Golgi-sulfotransferase genes CHST5 and GAL3ST2 of LS174T are determined by real-time quantitative RT-PCR. 3-FL, LNT2, and GOS-modulated LS174T gene expression profiles in a dose- and time-dependent manner. In addition, the upregulation of MUC2 is confirmed by immunofluorescence staining. Effects of 2'-FL, 3-FL, LNT2, and GOS on gene transcription of LS174T are also assessed during exposure to TNF-α, IL-13, or tunicamycin. During TNF-α challenge, 3-FL and LNT2 enhance MUC2 and TFF3 gene expression. After IL-13 exposure, 2'-FL, 3-FL, and LNT2 all show upregulating effects on MUC2; 3-FL and LNT2 also enhance TFF3 expression. LNT2 significantly reverses Tm-induced downregulation of TFF3, RETNLB, and CHST5. CONCLUSION: The findings indicate that hMOs may enhance mucus barrier function through direct modulation of intestinal goblet cells. Effects are structure- and stressor-dependent.

Modulation of Intestinal Epithelial Glycocalyx Development by Human Milk Oligosaccharides and Non-Digestible Carbohydrates.

SCOPE: The epithelial glycocalyx development is of great importance for microbial colonization. Human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) may modulate glycocalyx development. METHODS AND RESULTS: The effects of hMOs and NDCs on human gut epithelial cells (Caco2) are investigated by quantifying thickness and area coverage of adsorbed albumin, heparan sulfate (HS), and hyaluronic acid (HA) in the glycocalyx. Effects of hMOs (2'-FL and 3-FL) and NDCs [inulins with degrees of polymerization (DP) (DP3-DP10, DP10-DP60, DP30-DP60) and pectins with degrees of methylation (DM) (DM7, DM55, DM69)] are tested using immunofluorescence staining at 1 and 5 days stimulation. HMOs show a significant enhancing effect on glycocalyx development but effects are structure-dependent. 3-FL induces a stronger albumin adsorption and increases HS and HA stronger than 2'-FL. The DP3-DP10, DP30-60 inulins also increase glycocalyx development in a structure-dependent manner as DP3-DP10 selectively increases HS, while DP30-DP60 specifically increases HA. Pectins have less effects, and only increase albumin adsorption. CONCLUSION: Here, it is shown that 2'-FL and 3-FL and inulins stimulate glycocalyx development in a structure-dependent fashion. This may contribute to formulation of effective hMO and NDC formulations in infant formulas to support microbial colonization and gut barrier function.

Two-photon luminescence and second harmonic generation from gold micro-plates.

Micron-sized gold plates were prepared by reducing chloroauric acid with lemongrass extract. Their two-photon luminescence (TPL) and second harmonic generation (SHG) were investigated. The results show that the TPL and SHG intensity of gold plates is dependent on the wavelength and polarization of excitation laser. The TPL intensity of gold plates decreases with the increase of the excitation wavelength except for a small peak around 820-840 nm, while SHG intensity increases with the excitation wavelength redshift. In addition, it is found that the TPL intensity of the gold plate's edge is related with the angle between the edge orientation and the polarization direction of the excitation light. The TPL intensity increases with the angle increase from 0° to 90°.