Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal.

BACKGROUND: 5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. RESULTS: We develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by ß-glucosyltransferase (ß-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10-14 to 1 × 10-8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SIGNIFICANCE: This biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.

Apolipoprotein A-IV Has Bi-Functional Actions in Alcoholic Hepatitis by Regulating Hepatocyte Injury and Immune Cell Infiltration.

Alcohol abuse can lead to alcoholic hepatitis (AH), a worldwide public health issue with high morbidity and mortality. Here, we identified apolipoprotein A-IV (APOA4) as a biomarker and potential therapeutic target for AH. APOA4 expression was detected by Gene Expression Omnibus (GEO) databases, Immunohistochemistry, and qRT-PCR in AH. Bioinformatics Methods (protein-protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Set Enrichment Analysis (GSEA) were used to show down-stream gene and pathways of APOA4 in AH. AML-12 cells were used to evaluate the biological function of APOA4 using an ELISA kit (AST, ALT, and IL-1ß) and flow cytometry (ROS activity). Both in vivo and in vitro, APOA4 expression was significantly elevated in the AH model induced by alcohol (ETOH). AML-12 cell damage was specifically repaired by APOA4 deficiency, while AST, ALT, and IL-1ß activity that was increased by ETOH (200 µmol, 12 h) were suppressed. APOA4 inhibition increased intracellular ROS induced by ETOH, which was detected by flow cytometry. Functional and PPI network analyses showed Fcgamma receptor (FCGR) and platelet activation signaling were potential downstream pathways. We identified CIDEC as a downstream gene of APOA4. The CIDEC AUC values for the ROC curves were 0.861. At the same time, APOA4 silencing downregulated the expression of CIDEC, whereas the knockdown of CIDEC did not influence the expression of APOA4 in AML-12 cells. Collectively, APOA4 regulates CIDEC expression and immune cell infiltration and may hold great potential as a biomarker and therapeutic target for AH.

Effect of Alkylresorcinols on Autophagy, Migration, and Invasion of HepG2 Cells.

Alkylresorcinols are phenolic lipids that mainly exist in the cortex of grains, and they exhibit anticancer activity against various cancer cells in vitro. However, the underlying action mechanisms are still unclear. In our study, the influence of alkylresorcinols C19:0 and C21:0 (ARs) upon migration, invasion, and autophagy in human hepatoblastoma HepG2 cells was evaluated. Results showed that ARs at 80 and 160 µg/mL significantly suppressed cells proliferation, migration, and invasion, downregulated the expression of proteins RhoA and MMP-7 associated with migration and invasion. ARs at 160 µg/mL, the rate of LC3 puncta was appreciably increased. After autophagy was blocked by 3-MA or CQ, the expression of LC3II was significantly increased in 3-MA+ARs group and p62 was significantly decreased in CQ+ARs group. The results indicate that ARs may promote autophagic flow. ARs (80, 160 µg/mL) significantly inhibited the expression of proteins p-mTOR, p-PI3K, and p-Akt related to the PI3K/Akt pathway. The results of the present study suggest that ARs can activate autophagy and suppresses the biological behaviors of HepG2 cells by inhibiting the activation of MMP-7, Rho/Rho-associated protein kinase, and activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. PRACTICAL APPLICATION: The anticancer mechanism of ARs in wheat bran was studied, which provided a basis for the development of anticancer functional auxiliary food with wheat bran as raw material. It is of great practical significance to promote the effective utilization of grain processing by-products and improve the economic benefits of the grain industry.

Polyglycolic acid sutures embedded in abdominal acupoints for treatment of simple obesity in adults: a randomized control trial.

BACKGROUND: Acupoint catgut embedding therapy characterized by acupoint, needle and catgut are superior to traditional acupuncture, due to exerting more comprehensive therapeutic efficacy. However, it is still deficient in clinical evidence for polyglycolic acid sutures, a novel biodegradable material instead of catgut, embedded for the treatment of simple obesity. In our study, we investigate the efficacy and related mechanism of polyglycolic acid sutures embedded in abdominal acupoints on simple obese persons by a randomized control trial. METHODS: A total of 51 eligible participators were randomly allocated to a polyglycolic acid sutures embedding therapy (PASET) group (n = 28) or control group (n = 23). Participators in PASET group received polyglycolic acid sutures alternatively embedded in abdominal I group and II group acupoints in odd and even number therapeutic courses, and participators in control group were required to perform lifestyle modification. The duration of the study was 10 weeks. RESULTS: It suggested that PASET significantly reduced weight, body mass index, hip circumference, waist circumference, waist/hip ratio, waist-to-height ratio and thickness of abdominal subcutaneous fat tissue compared with those before treatment (p < 0.01), but lifestyle modification only illustrated downward trend of weight (p < 0.05). Moreover, PASET group also improved the evaluated scores in aspects of physical function, self-esteem, public distress and sexual life, as well as decreased blood pressure, glycemia, low density lipoprotein, uric acid and the levels of tumor necrosis factor-alpha, interleukin-1ß, and increased high density lipoprotein in comparison with those before treatment (p < 0.05), whose efficacies are superior to control group. Additionally, our results also indicate PASET is relative safe and its pain and discomfort can be tolerable. CONCLUSIONS: PASET distinctly ameliorates anthropometric data and quality of life in obese population, which associates with improvements of metabolic profile and inflammatory response. Based on the advantageous actions, we think PASET is an effective therapeutic approach to simple obesity treatment.Trial registration ChiCTR, ChiCTR1800015591. Registered 10 April 2018, http://www.chictr.org.cn/showproj.aspx?proj=23258.

[Precision genomic and translational medicine for acute myeloid leukemia].

Leukemia is a group of hematologic malignancy that has unfavorable prognosis and unclear mechanisms. In recent years, advances in leukemia research encompass the discovery of novel targets in acute myeloid leukemia drug resistance, epigenetic crosstalk in mixed lineage leukemia (MLL) leukemogenesis, genetic mechanisms of aggressive NK-cell leukemia, as well as the critical role of key epigenetic regulator in acute myeloid malignancy. Remarkably, researchers revealed that the histone modifying gene SETD2 as a new tumor suppressor and therapeutic target in patients with acute myeloid leukemia. Furthermore, low-dose chemotherapy as a frontline regiment in treating pediatric acute myeloid leukemia can substantially reduce the toxic side effects and treatment costs without impairing efficacy. Although advances in cancer genomics have greatly increased our understanding of the molecular characteristics in tumor biology, recent studies suggest that Darwinian evolution of intratumor heterogeneity represents a major challenge to develop therapeutic strategies to improve disease control. Researchers also dissected the distinct evolutionary dynamics under different chemotherapy regimens and the corresponding applications in the evaluation of treatment outcomes. Altogether, these efforts offered new opportunities for the development of acute myeloid leukemia diagnostics and therapeutics.

Thermal stability of ginkgolic acids from Ginkgo biloba and the effects of ginkgol C17:1 on the apoptosis and migration of SMMC7721 cells.

Ginkgolic acids are alkylsalicylic acid derivatives with a thermolabile carboxylic group from Ginkgo biloba L., and they exhibit anticancer activity. Their anticancer effects are closely associated with their thermal stability. In this study, the thermal decomposition of ginkgolic acids was analyzed at temperatures of 30, 50, 70 and 250°C. The results clearly showed that an obvious slow decarboxylation of the ginkgolic acids was detected at 70°C. When the temperature increased to 250°C, the decarboxylation reaction was rapidly completed. The ginkgolic acids were decarboxylated to yield ginkgols. The ginkgols C13:0, C15:1 and C17:1 were separated and definitively identified by IR, NMR and GC-MS. The cytotoxic effects of ginkgols C13:0, C15:1 and C17:1 were tested and compared with those of the corresponding ginkgolic acids. An MTT assay showed that ginkgol C17:1 (48-h IC50=8.5 µg·ml(-1)) has the strongest inhibition on SMMC-7721 cells in a dose- and time-dependent manner. The anticancer action may occur via the induction of apoptosis by the activation of caspases-3, the upregulation of Bax expression, and the inhibition migration of SMMC7721 cells. The results indicated that ginkgol C17:1 might be useful for the further development of a hepatocellular carcinoma preventive agent.

Adipose-derived mesenchymal stem cells ameliorate STZ-induced pancreas damage in type 1 diabetes.

OBJECTIVE: To investigate the possibility of adipose-derived mesenchymal stem cells (ADSC) in the treatment of type 1 diabetes (T1D). METHODS: ADSC were isolated from the adipotic tissue of abdomen in Sprague-Dawley rats (4-6 week-old,female) and expanded in vitro. Cells were then identified by testing their phenotypes through flow cytometry. Balb/c mice (8 week-old, male) were divided into 3 groups: T1D group, ADSC group and control group. Streptozocin (50 mg/kg·d) were injected intraperitoneally into mice of T1D group and ADSC group for 5 consecutive days to establish the T1D model. In ADSC group, ADSC were injected intravenously on day 3 of STZ injection. In control group, only PBS was injected. Fasting blood glucose (FGB) level was examined once a week. At the end of the 4th week, animals were killed. The pathological changes of islet were showed by histochemistry through hematoxylin-eosin staining (HE staining). ß cell insulin expression was detected by quantum dots immunofluorescence histochemistry. RESULTS: After ADSC administration, FGB levels decreased significantly from the second week. Whereas FGB levels in T1D group increased significantly and continuously during the experimental period. Moreover, ADSC effectively suppressed pancreatic islet damage induced by STZ and increased the expression of insulin protein in pancreatic ß cells. CONCLUSIONS: Intravenuously injected ADSC can prevent STZ induced ß-cell destruction and decrease blood glucose level.

[A method for induction of mouse CD8α(+);CD11b(+);jagged2(high);regulatory dendritic cells with mesenchymal stem cells in vitro].

AIM: To develop a method for induction of the mouse bone marrow-derived CD8α(+);CD11b(+);jagged2(high);regulatory dendritic cells (DCregs) using mesenchymal stem cells (MSCs) in vitro. METHODS: BALB/c(H-2(d);)mouse bone marrow cells (BMCs) were isolated and induced to CD8α(+);CD11c(+);CD11b(+);DCs (DCs) by 4 cytokines in vitro for 3 d. The DCs were selected by flow cytometry (FCM), and co-cultured with MSCs for 10 d to get DCregs. Immunophenotypes, cell cycle, Jagged1 and Jagged2 ligands of the Notch pathway of DCs were analyzed by FCM before and after co-culture. RESULTS: The novel DCs were transformed into DCregs successfully. The expressions of CD86, CD80, CD40, and MHC-II significantly decreased (P<0.05), while those of CD205, Jagged1 and Jagged2 obviously increased on DCregs (P<0.05). Meanwhile, the percentage of treated MSCs cells went up in G2 and S phases. CONCLUSION: MSCs co-cultured with DCs can induce the development of DCregs, which have immune tolerance-associated phenotypes and higher proliferation ability. This mechanism might be related to the up-regulated Jagged1 and Jagged2 expressions and the T-cell Notch pathway activation.

Elevated carbon dioxide and/or ozone concentrations induce hormonal changes in Pinus tabulaeformis.

We investigated endogenous plant hormones and needle growth in Pinus tabulaeformis plants grown in open-top chambers and exposed to ambient or elevated concentrations of carbon dioxide (CO(2)) and/or ozone (O(3)). Exposure to elevated CO(2) for 100 days significantly increased the change in fresh needle weight, indole-3-acetic acid (IAA), isopentenyl-adenosine (iPA), and dihydrozeatin riboside (DHZR) content. Abscisic acid (ABA) content decreased, and no effect was observed on zeatin riboside (ZR) content or changes in needle dry weight. The ratios of IAA/ABA and total cytokinins (CKs)/ABA [Formula: see text] were increased. Elevated O(3) significantly decreased IAA and ZR, and decreased the ratios of IAA/ABA and CKs/ABA. Ozone treatment increased ABA content but did not change iPA or DHZR content or change fresh or dry needle weights. The combination treatment significantly increased ABA content and the IAA/ABA ratio but decreased the total CKs/ABA ratio and had no effect on CKs or IAA content or change in fresh and dry needle weights. The results indicate that elevated CO(2) ameliorated the effects of elevated O(3) on tree growth.

[Detection of methylation levels of multi-genes by real-time PCR in patients with myelodysplastic syndrome].

OBJECTIVE: To analyze the promoter methylation levels of p15, CDH1, DAPK and HICI genes of patients with myelodysplastic syndrome (MDS) and explore the relationship between the level of methylation and clinical features. METHODS: DNA methylation levels of p15, CDH1, DAPK and HICI in peripheral blood (PB) or bone marrow (BM) samples from 52 MDS patients were detected by real-time quantitative PCR. The correlation of the methylation level with clinical features and hematological findings was analyzed. 38 de novo AML patients and 46 normal individuals served as controls. RESULTS: The methylation levels of p15, CDH1, DAPK and HICI were 16.23 ± 21.69, 6.59 ± 9.39, 0.14 ± 0.11 and 7.81 ± 9.70 in BM, and 14.96 ± 20.16, 6.00 ± 9.26, 0.12 ± 0.14 and 6.74 ± 9.72 in PB, respectively from 18 MDS patients, and the difference between BM and PB was not statistically significant (P > 0.05). The methylation levels of p15 (14.70 ± 18.17) and CDH1 (6.61 ± 8.79) genes in high risk (RAEBI/II) MDS were significantly higher than in low risk (RCMD/RARS/5q-, p15: 1.99 ± 1.59, CDH1: 1.23 ± 1.14 and RCMD, p15: 3.02 ± 3.42, CDH1:1.53 ± 2.06) MDS or control (p15: 1.69 ± 1.82, CDH1: 1.01 ± 1.12) (P < 0.05). The methylation levels of DAPK gene had no difference among subtypes of MDS, and that of HIC1 gene only differed between RAEB I/II (9.16 ± 11.95) and control (2.49 ± 2.26) (P = 0.042). The difference of methylation levels of p15, CDH1, DAPK and HICI in BM was statistically significant among subtypes of MDS (P = 0.001, 0.003, 0.039, 0.023, respectively). And so did of p15 and DAPK in PB (P = 0.013, 0.006, respectively). The methylation level of p15 and CDH1 was significantly correlated with IPSS classification and blasts percentage in BM. CONCLUSIONS: p15 and CDH1 genes are special hypermethylation genes in MDS. Methylation level of HIC1 gene showed an upward tendency from low risk to high risk MDS.

[Effect of [Gly14]-Humanin on Abeta(25-35)-induced PC12 cell apoptosis].

AIM: To investigate the effect of [Gly14]-Humanin overexpression on Abeta(25-35);-induced PC12 cell apoptosis. METHODS: Recombinant plasmid pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells by liposome method. The subclone cell lines were obtained by persistent G418 selection. The HNG gene expression of PC12 cells was detected by immunocytochemistry. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability was determined by MTT assay, and apoptosis rate was detected by using flow cytometric analysis. Hochest33258 staining was used to observe the morphological changes of cellular nuclei. RESULTS: PC12 cell lines stably expressing HNG gene was successfully selected. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability of PC12 cells overexpression HNG was significant elevated compared with empty plasmid transfected cells (P<0.05), and the apoptosis rate was lower significantly (P<0.05). By Hoechst 33258 staining, the nuclei of empty plasmid transfected PC12 cells exhibited highly condensed and fragmented nuclei morphology which was the typical characteristics of apoptosis, and the nuclei of PC12 cells overexpression HNG were round and homogeneously stained. CONCLUSION: Overexpression of HNG prevented the cell apoptosis induced by Abeta(25-35); in PC12 cells.

Localization and translocation of RhoA protein in the human gastric cancer cell line SGC-7901.

AIM: To elucidate the localization of RhoA in gastric SGC-7901 cancer cells and its translocation by lysophosphatidic acid (LPA) and/or 8-chlorophenylthio-cAMP (CPT-cAMP). METHODS: Immunofluorescence microscopy was used to determine the localization of RhoA. Western blotting was used to detect both endogenous and exogenous RhoA in different cellular compartments (membrane, cytosol, nucleus) and the translocation of RhoA following treatment with LPA, CPT-cAMP, or CPT-cAMP + LPA. RESULTS: Immunofluorescence staining revealed endogenous RhoA to be localized in the membrane, the cytosol, and the nucleus, and its precise localization within the nucleus to be the nucleolus. Western blotting identified both endogenous and exogenous RhoA within different cellular compartments (membrane, cytosol, nucleus, nucleolus). After stimulation with LPA, the amount of RhoA within membrane and nuclear extracts increased, while it decreased in the cytosol fractions. After treatment with CPT-cAMP the amount of RhoA within the membrane and the nuclear extracts decreased, while it increased within the cytosol fraction. Treatment with a combination of both substances led to a decrease in RhoA in the membrane and the nucleus but to an increase in the cytosol. CONCLUSION: In SGC-7901 cells RhoA was found to be localized within the membrane, the cytosol, and the nucleus. Within the nucleus its precise localization could be demonstrated to be the nucleolus. Stimulation with LPA caused a translocation of RhoA from the cytosol towards the membrane and the nucleus; treatment with CPT-cAMP caused the opposite effect. Furthermore, pre-treatment with CPT-cAMP was found to block the effect of LPA.

[Effect of WISp39 on proliferation, cell cycle and apoptosis of U937 cells].

To investigate the effect of a novel p21-modulating protein WISp39 on proliferation, apoptosis and cell cycle of leukemia cells, the plasmid pLenti6/V5-WISp39 was constructed and transfected into the human myelocytic leukemia cell line-U937 cells. The expression of WISp39 was detected by real-time PCR at 48 hours after transfection, proliferation of U937 cells assayed by CCK-8, apoptosis and cell cycle were determined by flow cytometry. The results showed that plasmid pLenti6/V5-WISp39 could readily enhance the expression of WISp39 in U937 cells. A significant growth inhibition (37.6%) was observed in cells tranfected with pLenti6/V5-WISp39, while the control plasmid pLenti6/V5-lacZ showed little effect on U937 growth. Further analysis revealed that pLenti6/V5-WISp39 did not show obvious apoptosis induction effect, but it could really regulate U937 proliferation via cell cycle modulation. Compared with pLenti6/V5-lacZ, pLenti6/V5-WISp39 resulted in increase of cells in G(0)/G(1) phase by 10% at 48 hours after transfection. It is concluded that the WISp39 gene has no significant apoptosis induction effect on leukemic cells, but it can increase cells at G(0)/G(1) phase via effect on cell cycle, thus inhibiting the U937 proliferation. This result means WISp39 gene can act as a negative modulator on tumour cells.

[Biological effects of immune serum from fusion protein of beta-amyloid peptide and HbcAg/MIR on AD transgeneic cells].

OBJECTIVE: To investigate the expression of fusion protein c-Abeta-c, a fusion protein from the beta amyloid peptide (Abeta) and the Hepatitis B core antigen/ Major immunodominant region (HbcAg/MIR), in the BL21/pET28 prokaryotic expression system and to immunize mice with the expressed fusion protein and evaluate the immunogenicity and biological effects of the serum in vitro. METHODS: The recombinant prokaryotic expression plasmid PET-28a /c-Abeta-c was constructed by molecular cloning technique and the c-Abeta-c fusion gene expression was induced in E. coli BL21 by isopropyl-1-thio-b-Dgalactopyranoside (IPTG). The expressed fusion protein was analyzed by SDS-PAGE. Intraperitoneal injection (i.p.) of the purified c-Abeta-c fusion protein was given to the BALB/c mice. The anti-Abeta effect of the immune serum was detected by indirect ELISA. The biological effect of the immune serum on Alzheimer's disease (AD) transgeneic cells was assessed by MTT assay and flow cytometer. RESULTS: The c-Abeta-c fusion protein was found in the sediment of the isolated bacteria. The expressed protein comprised more than 30% of the total proteins in the bacteria sediment. The anti-Abeta antibody in the serum of the immunized mice reached 1:16000. The antiserum reduced the cytotoxicity of Abeta peptide in the AD transgeneic cells and significantly decreased the apoptosis of cells. CONCLUSION: The c-Abeta-c fusion protein has good Abeta immunogenicity and the animal immune serum efficiently inhibits the cytotoxicity of Abeta peptide.

[Effect of CCL23/myeloid progenitor inhibitory factor 1 (MPIF-1) on the proliferation, apoptosis and differentiation of U937 cells].

CCL23 is a human CC chemokine with potential suppression effects on both human and murine myeloid progenitor cells both in vitro and in vivo, and only expressed and released by dendritic cells differentiated from monocytes in blood cells. However, recent study has shown that CCL23 was over-expressed in bone marrow and peripheral blood cells from pediatric patients with acute myeloid leukemia (AML). In order to investigate the effects of CCL23 on the development, therapy and prognosis of leukemia, the U937 cells, a leukemic cell strain, were adopted and cultured with rhCCL23 for 72 hours. The cell proliferation and apoptosis rate were detected by Cell Counting Kit-8 and FITC-AnnexinV/PI respectively; the morphologic changes and the expression of CCR1 (the only receptor of CCL23 known by now) were observed during the differentiation process. The results showed that no obvious effect on the proliferation, apoptosis and differentiation of U937 was found by using CCL23 alone (P > 0.05), but cultured in combination with CCL23 and PMA, the differentiation of U937 cells were promoted remarkably, during which the CCR1 expression increased (P < 0.05). It is concluded that CCL23 alone did not inhibit the proliferation and differentiation of U937, while its use in combination with PMA may possess synergistic effect on inducting differentiation of U937 through the increase of receptor CCR1 expression.

Experimental and density functional theory and ab initio Hartree-Fock study on the vibrational spectra of 2-(4-fluorobenzylideneamino)-3-mercaptopropanoic acid.

2-(4-Fluorobenzylideneamino)-3-mercaptopropanoic acid (4-FC) was synthesized through the reaction of 4-fluorobenzaldehyde and l-cysteine in refluxing EtOH. Its structure was verified by (1)H NMR, FT-IR and Raman. The ground-state geometries were optimized at B3LYP/6-31G**, B3LYP/6-31G*, HF/6-31G** and HF/6-31G* levels without symmetry constrains, respectively. The vibrational wavenumbers of 4-FC were calculated at same level. The scaled theoretical spectra using B3LYP methods, which are in a good agreement with the experimental ones, are superior to those using HF methods.

[Effects of long-term swimming exercise on iron status of rats].

AIM: To observe the effects of different periods of exercise on the iron status. METHODS: Female rats were randomly divided into 3-, 6-, 12-month swimming exercise groups and their corresponding sedentary groups. The hematological indices of iron status and the non-heme iron (NHI) and total NHI (TNHI) of the organs were determined at the end of the desired period. RESULTS: As compared with the corresponding sedentary groups, plasma iron and transferrin-iron saturation of three exercise groups were decreased without significant changes of blood hemoglobin and hematocrit. The NHI contents and TNHI of the liver, spleen and kidney were decreased. Although the NHI contents of the heart decreased, TNHI was not significantly changed. TNHI of the organs in both the exercised and sedentary rats were found to increase with age. CONCLUSION: The exercise-induced low iron status with depleted iron storage is similar to the iron-deficiency status, but it could not be explained using the hypothesis of iron deficiency. Both the NHI redistribution and the maintained iron storage suggests the adaptation of low iron status to exercise. Therefore, the so-called exercise-induced iron deficiency could not exist.