Nanomolar Hg²⁺ Detection Using β‑Lactoglobulin-Stabilized Fluorescent Gold Nanoclusters in Beverage and Biological Media

Owing to diverse functionalities and metal binding abilities, proteins have been proven to be promising ligands in the synthesis of gold nanoclusters (Au NCs). In this work, we explored β-lactoglobulin (β-Lg), a protein byproduct generated during cheese processing, as a biotemplate for fabrication of Au NCs by a facile and green method for the first time. The as-prepared Au NCs are water soluble and highly fluorescent and exhibit high sensitivity and selectivity for Hg²⁺ detection in aqueous solution. Interestingly, we found that the fluorescence of these Au NCs is stable either in a variety of complex matrixes or over a broad pH range (5.0–13.0) and therefore can be explored as a cell and animal imaging agent. More importantly, we demonstrated that the β-lactoglobulin-stabilized Au NCs (β-Lg–Au NCs) could serve as a sensor for the detection and quantification of Hg²⁺ in beverages, urine, and serum with high sensitivity

<i>Fpn1^Tek/Tek</i> mice have liver iron loading but decreased <i>Bmp6</i> and <i>Hamp1</i> expression.

<p>(A) Liver and spleen non-heme iron concentrations. (B) Serum iron concentration (SI, µg/dL) and percent Tf saturation (TS%). (C–E) Liver mRNA levels of <i>Hamp1</i> (C), <i>Bmp6</i> (D) and <i>Epo</i> (E). (F) Liver p-Smad1/5/8, Smad1, p-Erk1/2, Erk1/2, p-Stat3, Stat3 and β-actin protein levels were measured in 13–15-day-old male <i>Fpn1^flox/flox</i> and <i>Fpn1^Tek/Tek</i> mice (n = 6–7 per group), (G) Summary of the results in (F), quantitated using densitometry. Summary data are presented as mean ± SD. <b>*</b>P<0.05; <b>**</b>P<0.01.</p

Fine-Mapping and Genetic Analysis of the Loci Affecting Hepatic Iron Overload in Mice

<div><p>The liver, as the major organ for iron storage and production of hepcidin, plays pivotal roles in maintaining mammalian iron homeostasis. A previous study showed that Quantitative Trait Loci (QTLs) on chromosome 7 (Chr7) and 16 (Chr16) may control hepatic non-heme iron overload in an F2 intercross derived from C57BL/6J (B6) and SWR/J (SWR) mice. In this study, we aimed to validate the existence of these loci and identify the genes responsible for the phenotypic variations by generating congenic mice carrying SWR chromosome segments expanding these QTLs (D7Mit68-D7Mit71 and D16Mit125-D16Mit185, respectively). We excluded involvement of Chr7 based on the lack of iron accumulation in congenic mice. In contrast, liver iron accumulation was observed in Chr16 congenic mice. Through use of a series of subcongenic murine lines the interval on Chr16 was further fine-mapped to a 0.8 Mb segment spanning 11 genes. We found that the mRNA expression pattern in the liver remained unchanged for all 11 genes tested. Most importantly, we detected 4 missense mutations in 3 candidate genes including Sidt1 (P172R), Spice1(R708S), Boc (Q1051R) and Boc (S450-insertion in B6 allele) in the liver of SWR homozygous congenic mice. To further delineate potential modifier gene(s), we reconstituted seven candidate genes, <i>Sidt1</i>, <i>Boc</i>, <i>Zdhhc23</i>, <i>Gramd1c</i>, <i>Atp6v1a</i>, <i>Naa50</i> and <i>Gtpbp8</i>, in mouse liver through hydrodynamic transfection. However, we were unable to detect significant changes in liver iron levels upon reconstitution of these candidate genes. Taken together, our work provides strong genetic evidence of the existence of iron modifiers on Chr16. Moreover, we were able to delineate the phenotypically responsible region to a 0.8 Mb region containing 11 coding genes, 3 of which harbor missense mutations, using a series of congenic mice.</p></div

<i>Bmp6</i> and <i>Hamp1</i> expression decreases in the livers of <i>Fpn1^Alb/Alb</i> mice with high iron demand.

<p>(A) Liver and spleen non-heme iron concentrations. (B) SI and TS%. (C–E) Liver mRNA levels of <i>Hamp1</i> (C), <i>Bmp6</i> (D), <i>Epo</i> (E). and (F) Liver p-Smad1/5/8, Smad1, p-Erk1/2, Erk1/2, p-Stat3, Stat3 and β-actin protein levels were measured in 3-week-old male <i>Fpn1^flox/flox</i> and <i>Fpn1^Alb/Alb</i> mice fed an iron-rich diet for one week, and then transferred to an iron-deficient diet for one month (n = 5 per group). (G) Summary of the results in (F), quantitated using densitometry. Summary data are presented as mean ± SD. <b>*</b>P<0.05; <b>**</b>P<0.01.</p

Changes in serum Tf-bound iron levels are consistent with p-smad1/5/8 and <i>Hamp1</i> levels in <i>Fpn1^{Alb/Alb;LysM/LysM}</i> mice given an iron-deficient diet.

<p>(A) Time course of liver and spleen non-heme iron concentrations after switching to an iron-deficient diet. (B) Time course of SI and TS% levels. (C–E) Time course of liver mRNA levels of <i>Hamp1</i> (C), <i>Bmp6</i> (D), <i>Epo</i> (E). (F) Time course of liver p-Smad1/5/8, Smad1, p-Erk1/2, Erk1/2, p-Stat3, Stat3 and β-actin protein levels measured in 2-month-old male <i>Fpn1^{Alb/Alb;LysM/LysM}</i> mice fed an iron-deficient diet for 0, 2, 4 or 8 days (n = 5 mice per time point). (G) Summary of the results in (F), quantitated using densitometry. Summary data are presented as mean ± SD. <b>*</b>P<0.05; <b>**</b>P<0.01.</p

<i>Slc30a10</i> mutant zebrafish develop polycythemia, and chelation treatments improves the phenotype in mutant embryos.

<p>(A) Summary of the relative numbers of erythrocytes in 3-week-old and 1-week-old embryos (n = 6 sets of 50 embryos/group). (B and C) <i>Epo</i> expression was measured in 3-week-old and 1-week-old mutant zebrafish (B) and in the liver of adult zebrafish (C); n = 3 sets of 20 adults/group. (D and E) Mn-induced locomotor defects (D) and the darker color in liver (E) were rescued by treating embryos with EDTA-CaNa₂ (chelator, n = 3 sets of 20 embryos/group). (F-G) Example images of a Mn-exposed mutant embryo, showing a darker colored liver (F), and a Mn-exposed mutant embryo following EDTA-CaNa₂ treatment (G). (H) Following exposure to 300 μM Mn, mutant embryos were transferred to either Holt buffer alone or Holt buffer containing EDTA-CaNa₂, and locomotion was measured (n = 3 sets of 20 embryos/group). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001.</p

<i>Slc30a10</i> mutants develop impaired neurological function.

<p>(A) Example images of affected mutant adults and their unaffected siblings; note the thinner body shape of the affected siblings. (B) Summary of body weight of affected adults and unaffected siblings (n = 4 adults/group). (C) Affected mutants have higher Mn levels compared to their unaffected siblings (n = 4 adults/group). (D) Example movement traces of affected adults and their unaffected siblings. (E) Affected adults swim shorter distances than their unaffected siblings (n = 6 adults/group). (F) Summary of the reproductive capacity of WT and mutant adults (n = 20 adults/group). (G) Example images of a wild-type and mutant embryo, showing a darker color in the brain and liver (red arrows) in the mutant. (H) Mn exposure causes a larger increase in Mn accumulation in mutant animals compared to wild-type animals (n = 3 sets of 200 embryos/group). (I) Example images of two mutant embryos after Mn exposure. Note the distorted body shape of the Mn-treated mutant. (J) Locomotion (measured as swimming and a normal escape response) is reduced in mutant embryos following Mn treatment, whereas wild-type animals are not affected (n = 3 sets of 20 embryos/group). (K) Mutant embryos exposed to Mn swim a shorter distance than untreated mutants and wild-type embryos (n = 6 embryos/group). (L) <i>Slc32a1</i> expression is reduced in both wild-type and <i>slc30a10</i> embryos following Mn exposure (n = 3 sets of 20 embryos/group). (M-N) <i>Gabb1a</i> (M) and <i>gabb1b</i> (N) expression is reduced in Mn-exposed <i>slc30a10</i> mutants (n = 3 sets of 20 embryos/group). (O) Western blot analysis of Gad65/67 in wild-type and mutant embryos treated with or without Mn. (P-Q) <i>In situ</i> hybridization of <i>dat</i> and <i>th</i> in mutant embryos following Mn exposure, showing reduced expression of both genes compared to untreated mutants and WT embryos. (R) TUNEL staining in the brain of mutant embryos following Mn exposure, showing increased apoptosis compared to untreated mutants and WT embryos. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001; in K‒O, groups with different letters differed significantly (<i>p</i><0.05).</p

<i>Slc30a10</i> mutant zebrafish develop liver damage.

<p>(A) Oil red O staining shows that mutant embryos have a slightly fatty liver (red arrows), which was worsened upon exposure to Mn. (B-C) HE staining (B) and Oil red O staining (C) of frozen sections showing hepatic steatosis in mutant embryos following Mn exposure. (D) Example images of a wild-type and mutant embryo under Mn exposure, showing a darker colored liver in the mutant (red arrow). (E) Dose-response curve showing the percentage of embryos with liver color change versus Mn concentration (n = 3 sets of 20 embryos/group). (F) Image of the liver of a wild-type and mutant adult, showing severe fibrosis in the mutant liver with sirius red staining. (G-H) The fibrosis markers <i>col1a1a</i> and <i>ctgfa</i> were measured in the liver of both male and female WT and mutant animals (n = 3 sets of 20 adults/group; *<i>p</i><0.05 and **<i>p</i><0.01).</p

Slc30a10 functions as a Mn exporter in zebrafish.

<p>(A) <i>In situ</i> hybridization of wild-type zebrafish using the antisense <i>slc30a10</i> probe, showing expression in the brain and liver (red arrows), as well as the YSL (red arrowheads). A sense probe was used as a negative control. (B) Semi-quantitative RT-PCR of <i>slc30a10</i> mRNA in wild-type 1–7 dpf embryos, 14 dpf larvae, and 21 dpf larvae, showing the onset of expression at 5 dpf. (C) DNA and corresponding amino acid sequences of the wild-type (WT), 10-bp deletion (-10), and 1-bp insertion (+1) <i>slc30a10</i> alleles following CRISPR/Cas9-based editing. (D) Summary of <i>slc30a10</i> expression in WT and both <i>slc30a10</i> mutant lines (n = 3 sets of 50 embryos/group). (E-G) Mn, Fe, and Zn concentration was measured in wild-type and mutant 1-week-old embryos (E; n = 6 sets of 1000 embryos/group), 3-week-old larvae (F; n = 3 sets of 50 larvae/group), and 4-month-old adults (G; n = 3 adults/group). (H-J) Heterozygous embryos were exposed to the indicated concentrations of Mn²⁺ (H), Fe³⁺ (ferric ammonium citrate, FAC; I), or Zn²⁺ (J) for 24 hours at 5 dpf. At 6 dpf, <i>slc30a10</i> expression was measured; n = 3 sets of embryos/group. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001; N.S., not significant (<i>p</i>>0.05).</p

Functional crosstalk between SLC30A10 and ATP2C1 in HeLa cells.

<p>(A) HeLa cells were immunostained for ATP2C1 (green) and the Mn sensor protein GPP130 (blue). Following overnight exposure to 300 μM Mn, most of the ATP2C1 was translocated to the Golgi apparatus, and GPP130 was degraded. (B) HeLa cells were transfected with SLC30A10-Q308Stop-flag (Q308Stop, a mutant form of SLC30A10 in which codon 308 is changed to a stop codon); the cells were then immunostained for flag (red) and GPP130 (blue). Following Mn exposure, only half of the GPP130 was degraded. (C) Quantitative analysis of GPP130 immunoreactivity. (D) HeLa cells were transfected with galactosyltransferase-RFP (GalT) plasmid to label the Golgi apparatus (red); the cells were then immunostained for ATP2C1 (green). Following Mn exposure, most of the ATP2C1 was translocated to the Golgi apparatus, where it co-localized with GalT. (E-G) HeLa cells were co-transfected with the indicated constructs (E: GalT-RFP and SLC30A10-FLAG; F: GalT-RFP and SLC30A10-Q308Stop-flag; G: GalT-RFP and SLC30A10-1st-2nd-exons-deletion-flag, a mutant form of SLC30A10 in which the first and second exons are deleted); the cells were then immunostained for ATP2C1 (green) and FLAG (blue). (H) HeLa cells were mock-transfected cells or transfected with the indicated construct. After overnight exposure to Mn (500 μM), Mn concentration was measure using IPC-MS. ***<i>p</i><0.001.</p