Irregular particle morphology and membrane rupture facilitate ion gradients in the lumen of phagosomes.

Localized fluxes, production, and/or degradation coupled to limited diffusion are well known to result in stable spatial concentration gradients of biomolecules in the cell. In this study, we demonstrate that this also holds true for small ions, since we found that the close membrane apposition between the membrane of a phagosome and the surface of the cargo particle it encloses, together with localized membrane rupture, suffice for stable gradients of protons and iron cations within the lumen of the phagosome. Our data show that, in phagosomes containing hexapod-shaped silica colloid particles, the phagosomal membrane is ruptured at the positions of the tips of the rods, but not at other positions. This results in the confined leakage at these positions of protons and iron from the lumen of the phagosome into the cytosol. In contrast, acidification and iron accumulation still occur at the positions of the phagosomes nearer to the cores of the particles. Our study strengthens the concept that coupling metabolic and signaling reaction cascades can be spatially confined by localized limited diffusion.

Iron accelerates Fusobacterium nucleatum-induced CCL8 expression in macrophages and is associated with colorectal cancer progression.

Accumulating evidence suggests that high levels of Fusobacterium nucleatum in colorectal tumor tissues can be associated with poor prognosis in patients with colorectal cancer (CRC); however, data regarding distinct prognostic subgroups in F. nucleatum-positive CRC remain limited. Herein, we demonstrate that high-iron status was associated with a worse prognosis in patients with CRC with F. nucleatum. Patients with CRC presenting elevated serum transferrin saturation exhibited preferential iron deposition in macrophages in the tumor microenvironment. In addition, F. nucleatum induced CCL8 expression in macrophages via the TLR4/NF-κB signaling pathway, which was inhibited by iron deficiency. Mechanistically, iron attenuated the inhibitory phosphorylation of NF-κB p65 by activating serine/threonine phosphatases, augmenting tumor-promoting chemokine production in macrophages. Our observations indicate a key role for iron in modulating the NF-κB signaling pathway and suggest its prognostic potential as a determining factor for interpatient heterogeneity in F. nucleatum-positive CRC.

High-Throughput Screening for the Discovery of Iron Homeostasis Modulators Using an Extremely Sensitive Fluorescent Probe.

High-throughput methods for monitoring subcellular labile Fe(II) are important for conducting studies on iron homeostasis and for the discovery of potential drug candidates for the treatment of iron deficiency or overload. Herein, a highly sensitive and robust fluorescent probe for the detection of intracellular labile Fe(II) is described. The probe was designed through the rational optimization of the reactivity and responsiveness for an Fe(II)-induced fluorogenic reaction based on deoxygenation of an N-oxide, which was developed in-house. The probe is ready to use for a 96-well-plate-based high-content imaging of labile Fe(II) in living cells. Using this simple method, we were able to conduct high-throughput screening of a chemical library containing 3399 compounds. The compound lomofungin was identified as a potential drug candidate for the intracellular enhancement of labile Fe(II) via a novel mechanism in which the ferritin protein was downregulated.

A Golgi-targeting fluorescent probe for labile Fe(ii) to reveal an abnormal cellular iron distribution induced by dysfunction of VPS35.

Iron is involved in numerous physiologically essential processes in our body. However, excessive iron is a pathogenic factor in neurodegenerative diseases, causing aberrant oxidative stress. Divalent metal transporter 1 (DMT1) acts as a primary transporter of Fe(ii) ions. The intracellular delivery of DMT1 toward the cellular membrane via the trans-Golgi network during the endocytotic process is partially regulated by a retromer-mediated protein-sorting system comprising vacuolar protein-sorting proteins (VPSs). Thus, together with DMT1, the Golgi-apparatus acts as a hub organelle in the delivery system for intracellular Fe(ii) ions. Dysfunction of the VPS-relevant protein sorting system can induce the abnormal delivery of DMT1 toward lysosomes concomitantly with Fe(ii) ions. To explore this issue, we developed a fluorescent probe, Gol-SiRhoNox, for the Golgi-specific detection of Fe(ii) ions by integrating our original N-oxide-based Fe(ii)-specific chemical switch, a new Golgi-localizable chemical motif, and polarity-sensitive fluorogenic scaffold. Our synchronous imaging study using Gol-SiRhoNox and LysoRhoNox, a previously developed fluorescent probe for lysosomal Fe(ii), revealed that the intracellular distribution balance of Fe(ii) ions between the Golgi apparatus and lysosomes is normally Golgi-dominant, whereas the lysosome-specific elevation of Fe(ii) ions was observed in cells with induced dysfunction of VPS35, a member of the retromer complex. Treatment of cells with dysfunctional VPS35 with R55, a molecular chaperone, resulted in the restoration of the subcellular distribution of Fe(ii) ions to the Golgi-dominant state. These results indicate that the impairment of the DMT1 traffic machinery affects subcellular iron homeostasis, promoting Fe(ii) leakage at the Golgi and lysosomal accumulation of Fe(ii) through missorting of DMT1.

A mitochondria-targeted fluorescent probe for selective detection of mitochondrial labile Fe(ii).

Mitochondria are iron-rich organelles that are involved in the process of energy production through the electron-transporting system and heme synthesis. We developed a new mitochondria-targeted fluorescent probe, MtFluNox/Ac-MtFluNox, for Fe(ii) based on N-oxide chemistry, which we recently established as a Fe(ii)-selective fluorogenic switch. The deacetylated form MtFluNox showed a turn-on response towards Fe(ii) with high metal selectivity in cuvette experiments, and an imaging study using its cell-compatible analogue Ac-MtFluNox demonstrated mitochondria-specific fluorescence enhancement in response to Fe(ii) in living cells. Furthermore, the probe was able to detect endogenously accumulated Fe(ii) induced as a result of the inhibition of heme synthesis.

Fe(II) Ion Release during Endocytotic Uptake of Iron Visualized by a Membrane-Anchoring Fe(II) Fluorescent Probe.

Iron is an essential transition metal species for all living organisms and plays various physiologically important roles on the basis of its redox activity; accordingly, the disruption of iron homeostasis triggers oxidative stress and cellular damage. Therefore, cells have developed sophisticated iron-uptake machinery to acquire iron while protecting cells from uncontrolled oxidative damage during the uptake process. To examine the detailed mechanism of iron uptake while controlling the redox status, it is necessary to develop useful methods with redox state selectivity, sensitivity, and organelle specificity to monitor labile iron, which is weakly bound to subcellular ligands. Here, we report the development of Mem-RhoNox to monitor local Fe(II) at the surface of the plasma membrane of living cells. The redox state-selective fluorescence response of the probe relies on our recently developed N-oxide strategy, which is applicable to fluorophores with dialkylarylamine in their π-conjugation systems. Mem-RhoNox consists of the N-oxygenated rhodamine scaffold, which has two arms, both of which are tethered with palmitoyl groups as membrane-anchoring domains. In an aqueous buffer, Ac-RhoNox, a model compound of Mem-RhoNox, shows a fluorescence turn-on response to the Fe(II) redox state-selectively. An imaging study with Mem-RhoNox and its derivatives reveals that labile Fe(II) is transiently generated during the major iron-uptake pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox is capable of monitoring endosomal Fe(II) in primary cultured neurons during endocytotic uptake. This report is the first example that identifies the generation of Fe(II) over the course of cellular iron-uptake processes.

A universal fluorogenic switch for Fe(ii) ion based on N-oxide chemistry permits the visualization of intracellular redox equilibrium shift towards labile iron in hypoxic tumor cells.

Iron (Fe) species play a number of biologically and pathologically important roles. In particular, iron is a key element in oxygen sensing in living tissue where its metabolism is intimately linked with oxygen metabolism. Regulation of redox balance of labile iron species to prevent the generation of iron-catalyzed reactive oxygen species (ROS) is critical to survival. However, studies on the redox homeostasis of iron species are challenging because of a lack of a redox-state-specific detection method for iron, in particular, labile Fe2+. In this study, a universal fluorogenic switching system is established, which is responsive to Fe2+ ion based on a unique N-oxide chemistry in which dialkylarylamine N-oxide is selectively deoxygenized by Fe2+ to generate various fluorescent probes of Fe2+-CoNox-1 (blue), FluNox-1 (green), and SiRhoNox-1 (red). All the probes exhibited fluorescence enhancement against Fe2+ with high selectivity both in cuvette and in living cells. Among the probes, SiRhoNox-1 showed an excellent fluorescence response with respect to both reaction rate and off/on signal contrast. Imaging studies were performed showing the intracellular redox equilibrium shift towards labile iron in response to reduced oxygen tension in living cells and 3D tumor spheroids using SiRhoNox-1, and it was found that the hypoxia induction of labile Fe2+ is independent of iron uptake, hypoxia-induced signaling, and hypoxia-activated enzymes. The present studies demonstrate the feasibility of developing sensitive and specific fluorescent probes for Fe2+ with refined photophysical characteristics that enable their broad application in the study of iron in various physiological and pathological conditions.

Ovarian endometriosis-associated stromal cells reveal persistently high affinity for iron.

Ovarian endometriosis is a recognized risk for infertility and epithelial ovarian cancer, presumably due to iron overload resulting from repeated hemorrhage. To find a clue for early detection and prevention of ovarian endometriosis-associated cancer, it is mandatory to evaluate catalytic (labile) ferrous iron (catalytic Fe(II)) and to study iron manipulation in ovarian endometriotic lesions. By the use of tissues from women of ovarian endometriosis as well as endometrial tissue from women with and without endometriosis, we for the first time performed histological analysis and cellular detection of catalytic Fe(II) with a specific fluorescent probe (HMRhoNox-M), and further evaluated iron transport proteins in the human specimens and in co-culture experiments using immortalized human eutopic/ectopic endometrial stromal cells (ESCs) in the presence or absence of epithelial cells (EpCs). The amounts of catalytic Fe(II) were higher in ectopic endometrial stromal cells (ecESCs) than in normal eutopic endometrial stromal cells (n-euESCs) both in the tissues and in the corresponding immortalized ESCs. ecESCs exhibited higher transferrin receptor 1 expression both in vivo and in vitro and lower ferroportin expression in vivo than n-euESCs, leading to sustained iron uptake. In co-culture experiments of ESCs with iron-loaded EpCs, ecESCs received catalytic ferrous iron from EpCs, but n-euESCs did not. These data suggest that ecESC play a protective role for cancer-target epithelial cells by collecting excess iron, and that these characteristics are retained in the immortalized ecESCs.

Past sporting activity during growth induces greater bone mineral content and enhances bone geometry in young men and women.

We aimed to determine the effect of past sporting activity on bone mineral content (BMC), areal bone mineral density (aBMD) in the lumbar spine and proximal femur, and bone geometry of the mid femur in young men and women. We assessed 142 subjects, comprising 79 young men (21.2 ± 0.8 years) and 63 premenopausal young women (21.4 ± 0.6 years). The subjects were classified into three groups, two on the basis of the age of starting to participate in sport [elementary school starters (6-12 years), junior high school to university starters (13-22 years)], and the third group had no participation in sport. We measured BMC and aBMD by dual-energy X-ray absorptiometry (DXA) in the lumbar spine and proximal femur, and bone geometric characteristics of the mid femur by magnetic resonance imaging (MRI), and calculated the osteogenic index (OI) of previous sporting activity. The OI correlated significantly with many MRI-determined measures of bone geometry; DXA-measured BMC and aBMD were effective indicators of previous sporting activity in both sexes. The female elementary school starters had significantly greater femoral mid-diaphyseal perimeters (vs the no-sport group), bone cross-sectional area (vs the 13-22-year-old starters and the no-sport group), and maximum and minimum second moment of area at the mid-diaphysis point of the femur (vs the no-sport group). The OI is a proven practicable and useful index. DXA- and MRI-determined geometric characteristics showed that high-impact, weight-bearing exercise before and in early puberty induces greater total proximal femur BMC and enhances femoral mid-diaphyseal size and shape, and that these benefits persisted in young adult women.

A new class of high-contrast Fe(II) selective fluorescent probes based on spirocyclized scaffolds for visualization of intracellular labile iron delivered by transferrin.

Iron is an essential metal nutrient that plays physiologically and pathologically important roles in biological systems. However, studies on the trafficking, storage, and functions of iron itself in living samples have remained challenging due to the lack of efficient methods for monitoring labile intracellular iron. Herein, we report a new class of Fe(2+)-selective fluorescent probes based on the spirocyclization of hydroxymethylrhodamine and hydroxymethylrhodol scaffolds controlled by using our recently established N-oxide chemistry as a Fe(2+)-selective switch of fluorescence response. By suppressing the background signal, the spirocyclization strategy improved the turn-on rate dramatically, and reducing the size of the substituents of the N-oxide group enhanced the reaction rate against Fe(2+), compared with the first generation N-oxide based Fe(2+) probe, RhoNox-1. These new probes showed significant enhancements in the fluorescence signal against not only the exogenously loaded Fe(2+) but also the endogenous Fe(2+) levels. Furthermore, we succeeded in monitoring the accumulation of labile iron in the lysosome induced by transferrin-mediated endocytosis with a turn-on fluorescence response.

Diffractive properties of obstructed vector Laguerre-Gaussian beam under tight focusing condition.

Diffractive and focusing properties of vector Laguerre-Gaussian beams with obstacle are investigated under tight focusing conditions. Using vector diffraction theory, intensity and polarization distributions near the focus at different orthogonal planes are calculated and analyzed for vector Laguerre-Gaussian beams. It is observed that the beam is able to compensate the distortion produced by obstacles when the size of the obstacle is small. The structural changes in the polarization distribution are not the same in different orthogonal planes. The polarization characteristics of the beam show a significant change when the size of the obstacle is large. A comparative study of the focusing and diffractive properties of vector Laguerre-Gaussian and vector Bessel-Gaussian beams has also been performed.