High Ferritin Is Not Needed in Hemodialysis Patients: A Retrospective Study of Total Body Iron and Oral Iron Replacement Therapy.

In vivo iron levels can be adjusted through intestinal iron absorption to be maintained at a suitable level; however, optimal iron levels in hemodialysis (HD) patients are unclear. In this study, we investigated total body iron (TBI), calculated as the sum of red blood cell (RBC) iron and iron stores, during courses of low-dose oral iron replacement therapy, and evaluated in vivo iron sufficiency and its indicators in HD patients. We analyzed data on 105 courses of low-dose iron replacement therapy administered to 83 patients on maintenance HD over 7 months. We evaluated changes in TBI, RBC iron, and iron stores from the initiation of treatment to month 7 in two groups of patients, namely, iron-therapy responders and non-responders. TBI showed significant increases until month 4 and plateaued thereafter in iron-therapy responders, and tended to increase and then reached a similar plateau in non-responders (month 7: 1900 ± 447 vs. 1900 ± 408 mg). Steady-state TBI was strongly correlated with body surface area (y = 1628.6x - 791.91, R2 = 0.88, p < 0.001). We observed constant TBI during oral iron replacement therapy suggesting the activation of a "mucosal block". The results suggest that body surface area has utility for estimating the required TBI with regression equations.

Oral Iron Absorption of Ferric Citrate Hydrate and Hepcidin-25 in Hemodialysis Patients: A Prospective, Multicenter, Observational Riona-Oral Iron Absorption Trial.

Oral ferric citrate hydrate (FCH) is effective for iron deficiencies in hemodialysis patients; however, how iron balance in the body affects iron absorption in the intestinal tract remains unclear. This prospective observational study (Riona-Oral Iron Absorption Trial, R-OIAT, UMIN 000031406) was conducted at 42 hemodialysis centers in Japan, wherein 268 hemodialysis patients without inflammation were enrolled and treated with a fixed amount of FCH for 6 months. We assessed the predictive value of hepcidin-25 for iron absorption and iron shift between ferritin (FTN) and red blood cells (RBCs) following FCH therapy. Serum iron changes at 2 h (ΔFe2h) after FCH ingestion were evaluated as iron absorption. The primary outcome was the quantitative delineation of iron variables with respect to ΔFe2h, and the secondary outcome was the description of the predictors of the body's iron balance. Generalized estimating equations (GEEs) were used to identify the determinants of iron absorption during each phase of FCH treatment. ΔFe2h increased when hepcidin-25 and TSAT decreased (-0.459, -0.643 to -0.276, p = 0.000; -0.648, -1.099 to -0.197, p = 0.005, respectively) in GEEs. FTN increased when RBCs decreased (-1.392, -1.749 to -1.035, p = 0.000) and hepcidin-25 increased (0.297, 0.239 to 0.355, p = 0.000). Limiting erythropoiesis to maintain hemoglobin levels induces RBC reduction in hemodialysis patients, resulting in increased hepcidin-25 and FTN levels. Hepcidin-25 production may prompt an iron shift from RBC iron to FTN iron, inhibiting iron absorption even with continued FCH intake.

Changes of biomarkers for erythropoiesis, iron metabolism, and FGF23 by supplementation with roxadustat in patients on hemodialysis.

This study aimed to confirm changes in biomarkers of erythropoiesis and iron metabolism and serum fibroblast growth factor 23 (FGF-23) during darbepoetin-α treatment and then switching to the hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat. A total of 28 patients on hemodialysis who received weekly doses of darbepoetin-α were switched to roxadustat. Biomarkers for erythropoiesis and iron metabolism and intact and C-terminal FGF-23 were measured in blood samples collected before the HD session on days - 7 (darbepoetin-α injection), - 4, and - 2, and days 0 (switch to roxadustat treatment, three times weekly), 3, 5, 7, 14, 21, and 28. Erythropoietin and erythroferrone levels were elevated on day - 4 by darbepoetin-α injection and decreased to baseline levels at day 0. Levels of erythropoietin were not significantly increased by roxadustat supplementation, but erythroferrone levels were continuously elevated, similar to darbepoetin-α treatment. Hepcidin-25 and total iron binding capacity were significantly decreased or increased in patients treated with roxadustat compared with darbepoetin-α. Changes of intact and C-terminal FGF-23 levels were parallel to changes of phosphate levels during roxadustat treatment. However, the actual and percentage changes of intact FGF-23 and C-terminal FGF-23 in patients with low ferritin levels were greater than those in patients with high ferritin levels. Roxadustat might stimulate erythropoiesis by increasing iron usage through hepcidin-25, which was suppressed by erythroferrone in the physiological erythropoietin condition. Changes of intact FGF-23 and C-terminal FGF-23 levels might be affected by roxadustat in patients on hemodialysis, especially those with a low-iron condition.

Gentle, Massage-like, Head Stroking Provokes Salivary Oxytocin Release.

Context: The evidence is growing that oxytocin (OXT), a hypothalamic hormone, can induce parturition and lactation, modulate affiliative behavior, and regulate stress and energy metabolism. Although the physiological effects of massage aren't fully understood, massage may affect OXT release and facilitate adaptive responses to stressors. Objectives: This study intended to examine the effects of gentle, massage-like head, stroking to determine whether it could have a direct influence on the release of OXT. Design: The research team performed a preliminary study. Setting: The study was conducted at Kanazawa Medical University in Kahoku and Mizuho Hospital, Tsubata, Ishikawa, Japan. Participants: Participants were 14 volunteers from Mizuho Hospital. Intervention: The 14 recruited participants were assigned to the massage group and received gentle, massage-like, head stroking, which lasted 60 minutes. Seven of those participants were randomly recruited to become a control group that rested only, without massage, on a different day than the massage occurred. Outcome Measures: Participants' saliva for both groups was drawn at baseline and postintervention on the different days. Salivary OXT was assayed using a highly sensitive chemiluminescence immunoassay. Analyses were performed at baseline before the intervention and postintervention. Results: The OXT secretion increased significantly in the massage group unlike in the rest group, which had no change. Conclusions: Gentle, massage-like, head stroking is an effective method of releasing endogenous OXT. These findings open up the possibility of using endogenous OXT as an adjunct therapy in both clinical and research settings.

Threshold of Serum Ferritin to Discriminate against Those at Greater Risk of Platelet Increase during Treatment with Hypoxia-Inducible Factor Prolyl Hydroxylase Domain Inhibitor.

INTRODUCTION: Hypoxia-inducible factor prolyl hydroxylase domain inhibitors (HIF-PHI) are a new treatment for renal anemia. HIF-PHI is believed to increase iron usage to improve availability of iron for erythropoiesis. Therefore, there is concern that HIF-PHI might be prone to iron deficiency and that thrombosis might be induced by increased platelet and transferrin levels due to this iron deficiency. METHODS: Relationship of iron-related factors with platelet count (PLT) and total iron-binding capacity (TIBC; which reflects the transferrin level) were examined in 29 patients who were treated with darbepoetin alfa (DA) and then switched to roxadustat (Rox). To determine how changes in PLT and TIBC related to changes in iron-related factors, univariable and multivariable linear regression models were applied. To examine what iron-related factors on Day 0 influenced change in PLT, we used receiver operating characteristic (ROC) curves and logistic regression analysis for a rate of change in PLT ≤0% as the endpoint. Logistic regression analysis was performed with the reference group having serum ferritin (s-ft) or Transferrin saturation below the corresponding cutoff value (low vs. high). RESULTS: Multivariable analysis showed significant positive correlations between the rate of change in PLT and the change in s-ft and red blood cells (RBC) count {ß-coefficients; 0.40 [95% confidence interval (CI): 0.17-0.62], p = 0.001} (ß-coefficients; 30.45 [95% CI: 10.90-50.00], p = 0.004). The rate of change in TIBC was significantly positively correlated with only the change in RBC count. The ROC showed a significant cutoff value for s-ft of 77.2 ng/mL (sensitivity 63.6%, specificity 83.3%, area under the curve 0.76, 95% CI 0.55-0.96). Multivariable logistic regression also showed that only high s-ft was significantly elevated (9.46, 95% CI 1.42-63.30, p = 0.020). CONCLUSIONS: This study showed that changes in PLT were correlated with s-ft and amount of hematopoiesis. This suggests that an increase in PLT due to iron levels is less likely when s-ft is 77.2 ng/mL or higher at the time of switching from DA to Rox. In contrast, TIBC was only related to hematopoiesis in these patients. Control of s-ft before initiation of HIF-PHI treatment and gradual hematopoiesis might reduce the risk of thrombosis when switching from erythropoiesis-stimulating agents to HIF-PHI.

Novel Platform for Regulation of Extracellular Vesicles and Metabolites Secretion from Cells Using a Multi-Linkable Horizontal Co-Culture Plate.

Microfluidics is applied in biotechnology research via the creation of microfluidic channels and reaction vessels. Filters are considered to be able to simulate microfluidics. A typical example is the cell culture insert, which comprises two vessels connected by a filter. Cell culture inserts have been used for years to study cell-to-cell communication. These systems generally have a bucket-in-bucket structure and are hereafter referred to as a vertical-type co-culture plate (VTCP). However, VTCPs have several disadvantages, such as the inability to simultaneously observe samples in both containers and the inability of cell-to-cell communication through the filters at high cell densities. In this study, we developed a novel horizontal-type co-culture plate (HTCP) to overcome these disadvantages and confirm its performance. In addition, we clarified the migration characteristics of substances secreted from cells in horizontal co-culture vessels. It is generally assumed that less material is exchanged between the horizontal vessels. However, the extracellular vesicle (EV) transfer was found to be twice as high when using HTCP. Other merits include control of the degree of co-culture via the placement of cells. We believe that this novel HTCP container will facilitate research on cell-to-cell communication in various fields.

Comprehensive analysis of protein-expression changes specific to immunoglobulin G4-related disease.

BACKGROUND AND AIMS: Immunoglobulin 4 (IgG4)-related disease (IgG4-RD) is a lymphoproliferative disorder characterized by elevated serum IgG4 levels and tissue infiltration of IgG4-positive plasma cells. We analyzed the serum proteins, whose levels varied based on the disease state and treatment. MATERIALS AND METHODS: Serum proteins from patients with IgG4-related disease and healthy subjects were resolved using two-dimensional electrophoresis, silver-stained, and scanned. Alternatively, the proteins were labeled with Cy2, Cy3, and Cy5 before electrophoresis. The proteins, whose expression differed significantly between patients and healthy individuals, and between before and after steroid treatment, were identified and validated using enzyme-linked immunosorbent assays. RESULTS: Pre-treatment sera from patients with IgG4-related disease was characterized by increased levels of immunoglobulins such as IgG1, IgG4; inflammatory factors such as α-1 antitrypsin (A1AT); and proteins associated with immune system regulation such as clusterin and leucine-rich α-2-glycoprotein (LRG-1). The serum levels of A1AT, LRG-1 and clusterin, during treatment with prednisolone for up to 12 months revealed that LRG-1 levels were halved after 1 month of treatment, comparable to those in healthy subjects; LRG-1 levels remained normal until the end of treatment. CONCLUSION: LRG-1 could serve as a novel biomarker of IgG4-related diseases.

Protective Effects of Collagen Tripeptides in Human Aortic Endothelial Cells by Restoring ROS-Induced Transcriptional Repression.

Collagen tripeptide (CTP) is defined as a functional food material derived from collagenase digests of type I collagen and contains a high concentration of tripeptides with a Gly-X-Y sequence. CTP has several biological effects, including the acceleration of fracture healing, ameliorating osteoarthritis, and improving dryness and photoaging of the skin. Recently, an antiatherosclerotic effect of CTP has been reported, although its molecular mechanism is yet to be determined. In this study, we examined the effects of CTP on primary cultured human aortic endothelial cells (HAECs) under oxidative stress, because oxidative endothelial dysfunction is a trigger of atherosclerosis. DNA microarray and RT-qPCR analyses showed that CTP treatment recovered the downregulated expression of several genes, including the interleukin-3 receptor subunit alpha (IL3RA), which were suppressed by reactive oxygen species (ROS) treatment in HAECs. Furthermore, IL3RA knockdown significantly decreased the viability of HAECs compared with control cells. RT-qPCR analysis also showed that solute carrier 15 family peptide transporters, which are involved in CTP absorption into cells, were expressed in HAECs at levels more than comparable to those of a CTP-responsive human osteoblastic cell line. These results indicated that CTP exerts a protective effect for HAECs, at least in part, by regulating the recovery of ROS-induced transcriptional repression.

Hypoxia-inducible factor prolyl hydroxylase domain inhibitor may maintain hemoglobin synthesis at lower serum ferritin and transferrin saturation levels than darbepoetin alfa.

BACKGROUND: Hypoxia-inducible factor (HIF) prolyl hydroxylase domain inhibitors, which have recently become clinically available for treating renal anemia, are attracting attention for their novel mechanisms of action. METHODS: Relationships of reticulocyte hemoglobin content (CHr), which reflects recent Hb synthesis, with serum ferritin (s-ft) and transferrin saturation (TSAT) were examined in 30 patients on hemodialysis after switching from darbepoetin alfa (DA) to roxadustat (Rox). Iron deficiency was defined as CHr < 32.0 pg. Cutoff values of s-ft and TSAT were determined using receiver operating characteristic curves for the endpoint CHr ≥ 32.0 pg. Logistic analysis was performed with the reference group having s-ft or TSAT below the corresponding cutoff value (low vs high). RESULTS: With the endpoint CHr ≥ 32.0 pg on Day 0, cutoff values for s-ft and TSAT were respectively 49.7 ng/mL and 21.6% on Day 0 and 35.5 ng/mL and 16.2% on Day 28. With the endpoint CHr ≥ 32.0 pg on Day 28, cutoff values for s-ft and TSAT on Day 0 were 81.6 ng/mL and 23.9%, respectively. According to multivariable logistic analysis, the odds ratios of CHr ≥ 32.0 pg on Day 0 were significantly higher for high TSAT on Day 0 [34.7 (95% CI 2.42-131.0), p<0.003] and Day 28 [24.8 (95% CI 2.75-224.0), p = 0.004]. There were no significant differences by s-ft. Odd ratios of CHr ≥ 32.0 pg on Day 28 were also significantly higher for high s-ft on Day 0 [16.0 (95% CI 1.57-163.0), p = 0.019] and high TSAT on Day 0 [13.5 (95% CI 1.24-147.0), p<0.033]. CONCLUSIONS: Our results suggest Hb synthesis was maintained with lower TSAT and s-ft during Rox therapy compared with DA therapy. To avoid iron deficiency during the 4 weeks after switching DA to Rox, ideal s-ft and TSAT levels before the switch are 81.6 ng/mL and 23.9%, respectively.

Erythropoiesis stimulating agents are associated with serum fibroblast growth factor 23 metabolism in patients on hemodialysis.

BACKGROUND: This study aimed to determine associations among short- and long-acting erythropoiesis stimulating agents (ESAs), changes in serum fibroblast growth factor 23 (FGF23) and biomarkers of iron metabolism. METHODS: Among 108 patients on hemodialysis (HD), 44 received every 2 weeks or monthly doses of continuous erythropoiesis receptor activator (CERA), 31 received weekly doses of darbepoetin-α, 24 received three doses per week of epoetin-ß and 9 were not treated with an ESA. Intact and C-terminal FGF23 and transferrin saturation (TSAT), ferritin, erythroferrone and hepcidin 25 were measured in blood samples collected before the HD session at the end of the dialysis week (baseline, Day 0) and on Days 3, 5, 7 and 14 thereafter. RESULTS: Levels of ferritin, hepcidin 25 and erythroferrone as well as TSAT were significantly decreased or elevated in patients treated with CERA compared with other types of ESAs. Levels of C-terminal FGF23 increased in all groups during the observation period. Levels of intact FGF23 and ratios of intact FGF23 to C-terminal FGF23 gradually decreased between Days 3 and 7 in the CERA but not in the other groups. Multivariate models associated changes in hepcidin 25 and phosphate with those of intact FGF23. CONCLUSION: The long-acting ESA CERA might influence levels of intact FGF23 by increasing FGF23 cleavage in patients on HD in association with prolonged hepcidin 25 suppression.

Optimizing hepcidin measurement with a proficiency test framework and standardization improvement.

Objectives: Hepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization. Methods: We developed a pilot PT together with the Dutch EQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM. Results: The pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07±0.24 nmol/L. Conclusions: We established a framework for future PT to enable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.

A Hypoxia-Inducible Factor Stabilizer Improves Hematopoiesis and Iron Metabolism Early after Administration to Treat Anemia in Hemodialysis Patients.

Roxadustat (Rox), a hypoxia-inducible factor (HIF) stabilizer, is now available for the treatment of anemia in hemodialysis (HD) patients. To investigate hematopoietic effect and iron metabolism, this study involved 30 HD patients who were initially treated with darbepoetin (DA), a conventional erythropoietin-stimulating agent, and then switched to Rox. We measured erythrocyte, reticulocyte indices, and iron-related factors at every HD during the first two weeks after the treatment switch (Days 0-14) and again on Days 21 and 28. We measured erythropoietin (EPO) concentration every week and examined their changes from Day-0 values. The same variables were measured in 15 HD patients who continued DA at every HD for one week. Iron-related factors were also measured on Days 14 and 28. In the Rox group, hepcidin significantly decreased from Day 2. The reticulocyte hemoglobin content (CHr) significantly increased on Day 4, but decreased with a significant increase in reticulocyte count from Day 7. Log10(serum ferritin) significantly decreased after Day 11. Log10(EPO concentration) was lower at all time points. Compared with the DA group, the Rox group showed significant differences in all variables except CHr. These results suggest that Rox improves hematopoiesis and iron metabolism early after administration independent of EPO concentration.

Effect of cholecalciferol on serum hepcidin and parameters of anaemia and CKD-MBD among haemodialysis patients: a randomized clinical trial.

In this multicentre double-blind randomized clinical trial, we investigated the effects of oral cholecalciferol supplementation on serum hepcidin and parameters related to anaemia and CKD-MBD among haemodialysis patients. Participants were assigned in a 2:2:1:1 ratio to either (1) thrice-weekly 3,000-IU cholecalciferol, (2) once-monthly cholecalciferol (equivalent to 9,000 IU/week), (3) thrice-weekly placebo, or (4) once-monthly placebo. We also examined the effect modifications by selected single nucleotide polymorphisms in vitamin D-related genes. Out of 96 participants, 94 were available at Month 3, and 88 completed the 6-month study. After adjustment for baseline values, serum hepcidin levels were higher at Day 3 in the combined cholecalciferol (vs. placebo) group, but were lower at Month 6 with increased erythropoietin resistance. Cholecalciferol increased serum 1,25(OH)2D levels, resulting in a greater proportion of patients who reduced the dose of active vitamin D at Month 6 (31% vs. 10% in the placebo group). Cholecalciferol also suppressed intact PTH only among patients with severe vitamin D deficiency. In conclusion, cholecalciferol supplementation increases serum hepcidin-25 levels in the short term and may increase erythropoietin resistance in the long term among haemodialysis patients. Both thrice-weekly and once-monthly supplementation effectively increases serum 1,25(OH)2D levels, and hence, reduces active vitamin D drugs.Clinical Trial Registry: This study was registered at ClinicalTrials.gov and University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) as NCT02214563 (registration date: 12/08/2014) and UMIN000011786 (registration date: 15/08/2014), respectively (please refer to the links below). ClinicalTrials.gov: https://clinicaltrials.gov/ct2/show/record/NCT02214563 . UMIN-CTR: https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000017152&language=E .

The target hemoglobin content values of reticulocytes for efficient anemia improvement are achieved by low ferritin levels and moderate transferrin saturation: a retrospective observational study.

Objectives: The optimal iron level in hemodialysis (HD) patients remains unclear. The hemoglobin content of reticulocytes (CHr) is a sensitive indicator of iron used for hematopoiesis. To identify the optimal iron content for HD patients, we investigated the relation between CHr levels and iron status, as well as the levels of hepcidin, a main regulator of iron metabolism.Methods: This study enrolled 181 HD outpatients treated with recombinant human erythropoietin (rHuEPO). A sensitivity analysis, using a generalized linear regression model that included the interaction term, was applied to determine the correlations between levels of CHr and those of serum ferritin (s-ft), transferrin saturation (TSAT), and hepcidin.Results: The greatest changes in correlation coefficients for levels of s-ft and TSAT with CHr levels indicated optimal cut-off points of 50 ng/mL (≤50 ng/mL, r = 0.47 vs >50 ng/mL, r = 0.22) and 24% (≤24%, r = 0.58 vs >24%, r = 0.08), respectively. The correlation coefficient for levels of CHr and hepcidin showed that the optimal lower and upper cut-off points were 20 ng/mL (≤20 ng/mL, r = 0.52 vs >20 ng/mL, r = -0.01) and 70 ng/mL (≤70 ng/mL, r = 0.36 vs >70 ng/mL, r = -0.45), respectively.Discussion: This study indicates that the amount of iron in HD patients is sufficient for hematopoiesis under conditions of low s-ft and moderate TSAT levels. High levels of hepcidin could induce negative iron metabolism in hematopoiesis.Conclusion: Therefore, controlling hepcidin levels to within approximately 20-70 ng/mL may prevent iron deficiency and reduced Hb synthesis, and may thus facilitate effective iron utilization in hematopoiesis.

Differential Impacts of Intravenous Iron Administration and Iron-Containing Phosphate Binders on Serum Intact Fibroblast Growth Factor 23 Levels.

AIMS: This study assessed the impact of iron administration on serum fibroblast growth factor 23 (FGF23) levels. METHODS: Of 123 hemodialysis (HD) patients treated with erythropoiesis-stimulating agents, 22 received once-weekly intravenous iron and 17 received daily oral iron with iron-containing phosphate binders. Intact FGF23 and biomarkers of iron metabolism were measured from blood samples drawn before each HD session, at baseline and on days 3, 5, 7, and 14. RESULTS: Phosphate levels did not differ among the 3 groups during the 14-day period. Ferritin levels were significantly increased in both iron treatment groups compared with the non-iron treatment group, but changes in transferrin saturation levels were similar in the intravenous iron and non-iron groups. However, intact FGF23 levels were continuously higher in the intravenous iron group than those in the other groups. CONCLUSION: Intravenous iron administration may influence intact FGF23 levels in HD patients independently of phosphate and iron metabolism.

GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.

Glucocorticoids (GCs) potently induce T-cell apoptosis in a GC receptor (GR)-dependent manner and are used to control lymphocyte function in clinical practice. However, its downstream pathways remain controversial. Here, we showed that GC-induced transcript 1 (GLCCI1) is a novel downstream molecule of the GC-GR cascade that acts as an antiapoptotic mediator in thymic T cells. GLCCI1 was highly phosphorylated and colocalized with microtubules in GLCCI1-transfected human embryonic kidney QBI293A cells. GR-dependent up-regulation of GLCCI1 was associated with GC-induced proapoptotic events in a cultured thymocyte cell line. However, GLCCI1 knockdown in a thymocyte cell line led to apoptosis. Consistently, transgenic mice overexpressing human GLCCI1 displayed enlarged thymi that consisted of larger numbers of thymocytes. Further molecular characterization showed that GLCCI1 bound to both dynein light chain LC8-type 1 (LC8) and its functional kinase, p21-protein activated kinase 1 (PAK1), thereby inhibiting the kinase activity of PAK1 toward LC8 phosphorylation, a crucial event in apoptotic signaling. GLCCI1 induction facilitated LC8 dimer formation and reduced Bim expression. Thus, GLCCI1 is a candidate factor involved in apoptosis regulation of thymic T cells.-Kiuchi, Z., Nishibori, Y., Kutsuna, S., Kotani, M., Hada, I., Kimura, T., Fukutomi, T., Fukuhara, D., Ito-Nitta, N., Kudo, A., Takata, T., Ishigaki, Y., Tomosugi, N., Tanaka, H., Matsushima, S., Ogasawara, S., Hirayama, Y., Takematsu, H., Yan, K. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.

Immuno-detection of mRNA-binding protein complex in human cells under transmission electron microscopy.

Transit from the nuclear complex to the cytoplasm through the nuclear pore complex permits modification of mRNA, including processing such as splicing, capping, and polyadenylation, etc. At each of these events, mRNA interacts with various proteins to form mRNA-protein complex. Visualizing the mRNA is crucial for understanding the mechanisms underlying mRNA processing and elucidating its structure and recent advances in mRNA imaging allow detection of real-time mRNA localization in living cells. However, these techniques revealed only the location of mRNA but cannot visualize the conformation of mRNA-protein complex in cells. On the other hand, transmission electron microscopy has been used to visualize the structure of the Balbiani ring-derived large mRNA, but their observations were limited to the insect cells. In this study, we visualized the structure of mRNA-protein complex in human culture cells by using immuno-electron microscopy. Through immuno-detection, an mRNA exon junction binding complex Y14, and its binding protien Upf2, different gold particle patterns were imaged with transmission electron microscopy and analyzed. Characteristic linear and stacked particle orientation were observed. Across the nuclear membrane, only linear aggregation pattern was observed, whereas the stacked aggregation pattern was detected in the cytoplasm. Our method is able to visualize mRNA-conformation and applicable to many cell types, including mammalian cells, where genes can easily be manipulated.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

[Corrigendum] Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm.

Following the publication of this article, after having re-examined our manuscript, an error was noted in the Acknowledgments section regarding the funding of our study. The Acknowledgments section should have appeared as follows: "This work was supported by grants from the Kanazawa Medical University (nos. C2014-3, C2015-1 and S2014-15), a grant from the Strategic Research Project (no. H2012-16 [S1201022]) from Kanazawa Medical University, Ministry of Education, Culture, Sports, Science and Technology, Japan (KAKENHI no. #25460376) and from the Sumitomo Science Foundation in Japan. We apologize to our funders for this error, and regret any confusion this may have caused. [the original article was published in the Molecular Medicine Reports 14: 655-660, 2016; DOI: 10.3892/mmr.2016.5331].