Nrf2 expands the intracellular pool of the chaperone AHSP in a cellular model of ß-thalassemia.

In ß-thalassemia, free α-globin chains are unstable and tend to aggregate or degrade, releasing toxic heme, porphyrins and iron, which produce reactive oxygen species (ROS). α-Hemoglobin-stabilizing protein (AHSP) is a potential modifier of ß-thalassemia due to its ability to escort free α-globin and inhibit the cellular production of ROS. The influence of AHSP on the redox equilibrium raises the question of whether AHSP expression is regulated by components of ROS signaling pathways and/or canonical redox proteins. Here, we report that AHSP expression in K562 cells could be stimulated by NFE2-related factor 2 (Nrf2) and its agonist tert-butylhydroquinone (tBHQ). This tBHQ-induced increase in AHSP expression was also observed in Ter119+ mouse erythroblasts at each individual stage during terminal erythroid differentiation. We further report that the AHSP level was elevated in α-globin-overexpressing K562 cells and staged erythroblasts from ßIVS-2-654 thalassemic mice. tBHQ treatment partially alleviated, whereas Nrf2 or AHSP knockdown exacerbated, α-globin precipitation and ROS production in fetal liver-derived thalassemic erythroid cells. MafG and Nrf2 occupancy at the MARE-1 site downstream of the AHSP transcription start site was detected in K562 cells. Finally, we show that MafG facilitated the activation of the AHSP gene in K562 cells by Nrf2. Our results demonstrate Nrf2-mediated feedback regulation of AHSP in response to excess α-globin, as occurs in ß-thalassemia.

Sirt6 regulates efficiency of mouse somatic reprogramming and maintenance of pluripotency.

BACKGROUND: Mouse somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. It has been reported that Sirtuin 6 (Sirt6), a member of the sirtuin family of NAD+-dependent protein deacetylases, is involved in embryonic stem cell differentiation. However, whether and how Sirt6 influences epigenetic reprogramming remains unknown. METHODS: Mouse embryonic fibroblasts isolated from transgenic Oct4-GFP reporter mice with or without Sirt6 were used for reprogramming by Yamanaka factors. Alkaline phosphatase-positive and OCT4-GFP-positive colony were counted to calculate reprogramming efficiency. OP9 feeder cell co-culture system was used to measure the hematopoietic differentiation from mouse ES and iPS cells. RNA sequencing was measured to identify the differential expressed genes due to loss of Sirt6 in somatic and pluripotent cells. RESULTS: In this study, we provide evidence that Sirt6 is involved in mouse somatic reprogramming. We found that loss of function of Sirt6 could significantly decrease reprogramming efficiency. Furthermore, we showed that Sirt6-null iPS-like cell line has intrinsically a differentiation defect even though the establishment of normal self-renewal. Particularly, by performing transcriptome analysis, we observed that several pluripotent transcriptional factors increase in knockout cell line, which explains the underlying loss of pluripotency in Sirt6-null iPS-like cell line. CONCLUSIONS: Taken together, we have identified a new regulatory role of Sirt6 in reprogramming and maintenance of pluripotency.

Curcumin and its promise as an anticancer drug: An analysis of its anticancer and antifungal effects in cancer and associated complications from invasive fungal infections.

Invasive fungal infections (IFI) are important complications of cancer, and they have become a major cause of morbidity and mortality in cancer patients. Effective anti-infection therapy is necessary to inhibit significant deterioration from these infections. However, they are difficult to treat, and increasing antifungal drug resistance often leads to a relapse. Curcumin, a natural component that is isolated from the rhizome of Curcuma longa plants, has attracted great interest among many scientists studying solid cancers over the last half century. Interestingly, curcumin provides an ideal alternative to current therapies because of its relatively safe profile, even at high doses. To date, curcumin's potent antifungal activity against different strains of Candida, Cryptococcus, Aspergillus, Trichosporon and Paracoccidioides have been reported, indicating that curcumin anticancer drugs may also possess an antifungal role, helping cancer patients to resist IFI complications. The aim of this review is to discuss curcumin's dual pharmacological activities regarding its applications as a natural anticancer and antifungal agent. These dual pharmacological activities are expected to lead to clinical trials and to improve infection survival among cancer patients.

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in ß-thalassemia-derived iPSCs.

ß-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the ß-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

Sox2 Deacetylation by Sirt1 Is Involved in Mouse Somatic Reprogramming.

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. Together with Oct4, Sox2 plays a major role as a master endogenous pluripotent genes trigger in reprogramming. It has been reported that Sirtuin 1 (Sirt1), a member of the Sirtuin family of NAD(+) -dependent protein deacetylases, is involved in embryonic stem cell antioxidation, differentiation, and individual development. However, as a deacetylation enzyme, whether Sirt1 influences reprogramming through its post-translational modification function remains unknown. In this study, we provide evidence that deacetylation of Sox2 by Sirt1 is required for reprogramming. We found that a low level of Sox2 acetylation could significantly increase reprogramming efficiency. Furthermore, we found that Sox2 can be deacetylated by Sirt1 in an Oct4-mediated manner. Compared with wild-type cells, Sirt1-null mouse embryonic fibroblasts exhibit decreased reprogramming efficiency, and overexpression of Sirt1 rescues this defect. In addition, Sirt1 functions in the regulation of reprogramming through deacetylating Sox2. Taken together, we have identified a new regulatory role of Sirt1 in reprogramming and provided a link between deacetylation events and somatic cell reprogramming. Stem Cells 2015;33:2135-2147.

Anti-ageing effects of protocatechuic acid from Alpinia on spleen and liver antioxidative system of senescent mice.

The effects of Alpinia protocatechuic acid (PCA) on spleen and liver antioxidant system in aged rats have been studied. Alpinia PCA, a phenolic compound, was first isolated from the dried fruits of Alpinia Oxyphylla Miq. in our laboratory. Young and aged rats were injected intraperitoneally with Alpinia PCA at single doses of 5 mg kg(-1) (low dose) or 10 mg kg(-1) (high dose) per day for 7 days. The activities of endogenous antioxidants and the content of lipid peroxide in spleen and liver were assayed. Compared with young group, aged rats had significantly lower splenic weights, lower activities of glutathione peroxidase (GSH-PX) and catalase (CAT), higher level of malondialdehyde (MDA) in spleen and liver. The results proved that Alpinia PCA significantly elevated the splenic weights, increased the activities of GSH-PX and CAT and decreased the MDA level of aged rats. All these suggested that Alpinia PCA was a potential anti-ageing agent, and its effects on spleen and liver were achieved at least partly by promoting endogenous antioxidant enzymatic activities and normalizing age-associated alterations. It may be therapeutically useful to minimize age-associated disorders where oxidative damage is the major cause.

[Condition of Epstein Barr virus infection in hospitalized children from Wuhan region].

OBJECTIVE: To study the condition and clinical characteristics of Epstein Barr virus (EBV) infection in hospitalized children from Wuhan region. METHODS: A total of 14 840 hospitalized children were classified into five age groups: less than 6 months old, 6 months to 1 year old, 1 to 3 years old, 3 to 7 years old and 7 to 15 years old. The antibodies IgM and IgG to EBV capsid antigen (VCA) were detected using ELISA. RESULTS: In the 14 840 hospitalized children, 7 899 were positive for EBV antibodies, with an infection rate of 53.23%. The positive rate of VCA-IgM was 4.05% (601/14 840) and that of VCA-IgG was 49.18% (7 298/14 840). The lowest positive rate of VCA-IgM (0.11%) was found in the group of less than 6 months old and the highest positive rate of VCA-IgG (79.83%) was found in the group of 7 to 15 years old. Of the 601 children with positive VCA-IgM, 429 (71.4%) suffered from respiratory tract infection. CONCLUSIONS: EBV infection is very common in hospitalized children in Wuhan. Respiratory tract infection is a leading disease in children with positive EBV antibodies. The infection rate of EBV in different age groups is different.

Improved preservation of human red blood cells by lyophilization.

The lyophilization of human red blood cells has important implications for blood transfusion in clinical medicine. In this study, sugars, human serum albumin, polyvinylpyrrolidone, and dimethyl sulfoxide were used as protective reagents for the lyophilization of red blood cells. Freezing temperature, shelf temperature, and the rehydration conditions were optimized. The results showed that extracellular disaccharides, especially trehalose, did not increase the recovery of hemoglobin. However, when the concentration of human serum albumin was higher than 25%, it had a considerable protective effect on the recovery of lyophilized red blood cells; the cellular hemoglobin recovery was over 70%, which was significantly higher than that in the group without human serum albumin (P<0.01). As the concentration of polyvinylpyrrolidone was increased, the extent of vitrification also increased. But when the concentration of polyvinylpyrrolidone was over 40%, the resulting concentration of free hemoglobin was over 1g/L, which was significantly higher than that with 40% (P<0.01). When lyophilization was carried out after freezing at different temperatures, the recovery of cells and hemoglobin was 70-80% and there were no significant differences among the five groups. When the shelf temperature was higher than -30 degrees C, the samples were partly collapsed, but when the shelf temperature was lower than -30 degrees C, the recovery of cells in the -40 and -45 degrees C groups was significantly higher than in the -30 and -35 degrees C groups (P<0.05). The recovery of cells and hemoglobin after lyophilization and rehydration in solutions containing low concentrations of polymers was over 80%, which is significantly higher than the other groups (P<0.01). In addition, when the temperature was higher than 25 degrees C, the concentration of free hemoglobin was significantly lower than it was at 4 degrees C (P<0.01). In conclusion, our study showed the lyophilization of red blood cells is feasible. Disaccharides have no protective effect on lyophilized cells when they are only extracellular and extensive vitrification may be not beneficial. Although the recovery of cells after lyophilization and rehydration by our method was over 70%, the ultrastructure of the cells may be compromised and some hemolysis does still exist. Further research is required.

[Effect of pre-freezing temperature and lyophilizer shelf temperature on recovery of red blood cells after lyophilization].

To study effect of pre-freezing temperature and lyophilizer shelf temperature on recovery of human red blood cells after lyophilization and determine solidifying temperature of this lyophilization system, the protective solution composed of 7% DMSO, 40% polyvinylpyrrolidone (PVP) and isotonic buffer were adopted to lyophilize red blood cells at different pre-freezing temperatures or shelf temperatures. At first, fresh whole blood was centrifugated, washed and equilibrized to prepare concentrated red blood cells. Then concentrated red blood cells were mixed with the protective solution at 1:3 and pre-freezed at different temperature (-20, -35, -45, -80 or -196 degrees C) before lyophilization in lyophilizer. To study effect of shelf temperature on lyophilization of red blood cells, red blood cells were lyophilized at different shelf temperature after pre-freeze at -80 degrees C. After lyophilization, the samples were quickly rehydrated by 37 degrees C rehydration solution. The results showed the recovery rate of red blood cells and hemoglobin after pre-freeze at different temperature and lyophilization were > 85% and > 75%, there was not significant difference among these groups, but the concentration of free hemoglobin in -196 degrees C group was significantly higher than that in other groups (P < 0.01). With decreasing of shelf temperature, the lyophilizing time was also prolonged. When shelf temperature was > or = -25 degrees C, samples were not fully lyophilized; when shelf temperature was < or = -30 degrees C, the recovery rate of red blood cells and hemoglobin after lyophilization and rehydration were above 90%; after washed to isotonic state, the recovery rate of hemoglobin of the four groups was similar to each other. In conclusion, only when pre-freezing temperature is between -20 and -80 degrees C and the lyophilizer shelf temperature is < or = -30 degrees C, the effect of lyophilization is better, but the effect of excessively low pre-freezing temperature may even be worse.

[Dynamic changes of ultrastructure of erythrocytes in prolonged preservation at 4 degrees C].

To study the dynamic changes of ultrastructure of erythrocytes in prolonged preservation of blood with preservative fluid containing superoxide dismutase (SOD), the whole blood samples were preserved at 4 degrees C in SOD-containing solution, the morphologic changes of erythrocyte were dynamically ob served by transmission microscopy after preservation for 42, 75 and 85 days, an d the blood samples preserved in GMA solution served as control. Three variance was applied to analyze the data with SAS software. The results showed that the metamorphotic rates of erythrocyte preserved in SOD-containing solution for 42, 75 and 85 days were lower than those of erythrocytes preserved in GMA solution. Most of metamorphotic rates of erythrocyte preserved in SOD-containing solution for 42, 75 and 85 days were correspond to those of erythrocytes preserved in GMA solution for 42 days, or even lower. It is concluded that SOD-containing preservative fluid might help to maintain the normal morphology of erythrocytes in prolonged preservation at 4 degrees C.

[Effect of vitrification state of protective solutions on recovery of red blood cells after lyophilization preservation].

To study effect of vitrification state of protective solutions on recovery of red blood cells after lyophilization, four protective solutions composed of isotonic buffers containing 7% DMSO (v/v) and 20%, 30%, 40% or 50% polyvinylpyrrolidone (PVP) (w/v) were adopted. Vitrification state of protective solutions was examined first when white ice crystal appeared in any protective solution during freezing or thawing, if the used solution was not a vitrification solution. Red blood cells were lyophilized in MINILYO45 freeze-dryer after washing, mixing with protective solutions and prefreezing. After lyophilization, the samples were quickly rehydrated by 37 degrees C rehydration solution. The results showed that in vitrification and devitrification experiments, white ice crystal appeared in solution of 20% PVP + 7% DMSO and 30% PVP + 7% DMSO during freezing and thawing; vitrification appeared in solution of 40% PVP + 7% DMSO during freezing, but devitrification appeared during thawing; vitrification appeared in solution of 50% PVP + 7% DMSO during freezing and thawing. After rehydration, the recoveries of red blood cells and hemoglobin in 40% PVP + 7% DMSO group were (81.36 +/- 14.94)% and (77.54 +/- 12.86)%, which were significantly higher than that in 20% PVP + 7% DMSO, 30% PVP + 7% DMSO and 50% PVP + 7% DMSO groups (P < 0.01). The concentration of free hemoglobin in 40% PVP + 7% DMSO group was also significantly lower than that in other three groups (P < 0.01). With increase of PVP concentration in protective solutions, vitrification state and protective effect of these solutions also increased; when concentration of PVP in protective solution was 40% though it was not a vitrification solution, the effect of lyophilization was the best; but when concentration of PVP further increased to 50%, though it was a vitrification solution, the effect decreased. It is concluded that excessive vitrification state could not benefit lyophilization of red blood cells.

[Analysis on ultrastructure of lyophilized red blood cells].

The objective of the present study was designed to evaluate lyophilized red blood cells of the ultrastructure. Blood was drawn from healthy adult. In group 1, sample was fresh blood; in group 2, sample was added 35% glycerine, stored at -80 degrees C for 24 hours; in group 3, red blood cells stored at 4 degrees C for 5 hours, then were lyophilized for 16 hour. The sample was resuspended for measurements of count and electron microscopy study. The result showed that lyophilized red blood cells possessed relative integrated structure, red blood cell recovery was 53%. The mean diameter, optical density and integral optical density of red blood cell were 4.7 +/- 0.4, 0.14 +/- 0.03 and 1.58 +/- 0.46 in group 1; 4.6 +/- 0.7, 0.14 +/- 0.02 and 2.35 +/- 0.64 in group 2; 4.4 +/- 0.4, 0.17 +/- 0.05 and 2.35 +/- 0.46 in group 3, respectively. There was no significant difference in lyophilized and frozen group, but there was significant difference in lyophilized group and normal group. In conclusion, human red blood cells could be successfully lyophilized and possess relative integrated structure. The mean diameter, optical density and integral optical density of lyophilized red blood cells were similar to that of cryopreservation red cells.

[Labeling and survival studies on rabbit RBC].

AIM: To establish a simple and effective method for RBCs labeling and survival assays, and the qualities of rabbit RBCs preserved in GMA solution at 4 degrees C were verified. METHODS: The bloods were taken through the ear arteries of the rabbits. The RBCs were labeled by fluorescein isothiocyanate (FITC), and were reinjected to the same rabbit through ear veins. The percentage of FITC labeled RBCs was assayed by FACS at a series of times after injection. The SAS software was employed to analyze the data and establish the regression equations. The 24-hour recovery and the half-life span of the labeled RBCs were calculated according to the equations. RESULTS: The 24-hour recovery and the half-life span of the labeled RBCs in the control group were 93.76% +/- 5.40% and 22.50% +/- 4.37 days respectively, which was in agreement with the previous papers. The 24-hour recovery and the half-life span of the labeled RBCs in the GMA group were 89.13% +/- 7.10% and 11.41% +/- 1.63 days respectively, which was coincident with the infusion conditions. CONCLUSION: Compared with other methods of RBCs labeling in vivo, FITC labeling was thought to be easier and cheaper to use, which could facilitate the analysis of the biological character of the labeled cells, and could be used to trace the fate of labeled cells.

[A simple and convenient method for assaying human RBCs glycolysis rate without oxygen].

AIM: To convenience of the methods for assaying red blood cell glycolysis without oxygen condition in the studies. METHODS: Reagent kit of glucose, perchloric acid, visible light prismatic photometer, battle of nitrogen and rocking bed are used in the studies. The process includes 4 steps prepare Tris- HCI solution and so on, assay of red blood cell glycolysis without oxygen condition and account of glycolysis rate. RESULTS: Human red blood cells stored at 4 degrees C for 75 d, in SOD solution, the glycolysis rate is 86.2% +/- 5.0%, distinctly better than GMA solution (39.2% +/- 8.9%). CONCLUSION: The methods of assaying glycolysis without oxygen condition not use Habea's apparatus. The operation is convenient and simple and its determinations can be performed in ordinary laboratory and is is accurate.

[Studies of protective effect of SOD on damage of human RBCs stored at 4 degrees C].

AIM: To investigate the mechanism of protective effect of SOD (superoxide dismutase) on damage of RBCs stored at 4 degrees C, the studies of erythrocyte glucose and energy metabolism were performed. METHODS: whole blood collected from healthy donors and stored at 4 degrees C in ACD, GMA and SOD solutions. Before and post storage, some parameters were assayed. Standard methods were used for the in vitro tests. The 24-hour in vivo recoveries were measured by FTTC (Fluorescein 5-isothiocyanate) from SIGMA Company. RESULTS: All parameters of red blood cell glucolysis rate without oxygen condition, ATP, PK (pyruvic kinase) and 24 h recoveries level were 86.2%, 56.4%, 64.3% and 86.2% of normal respectively stored in SOD solution at 4 degrees C for 75 days, distinctly more than in ACD and GMA groups at 75 days stored. The 24 h recovery at 75d in group SOD was near the recovery at 42d in group GMA. CONCLUSION: Whole blood in SOD solution can be stored satisfactorily for 75 days at 4 degrees C, and furnished theoretical evidence for RBCs survival.

[New tactics of human red blood cells stored at 4 degrees C-protective effect of antioxidant solution on red blood cells damage].

Aliquots of venous blood from healthy donor were collected in plastic blood storage bags with ACD, GMA or antioxidant solution (superoxide dismutase, SOD), respectively, and stored at 4 degrees C. After storage for varying periods, the parameters of the blood were detected in the blood samples. Results showed that the parameters of the blood stored at 4 degrees C for 75 days in SOD group were following: the recovery of RBC-Hb was 87.2%, plasma-Hb (mg/L) was 193.2, P50 (mmHg) was 34.0 (normal value was 33.1); deformability (DImax) was 0.2413 (74.3% of normal value). There was no evident hemolysis, color change, air bubble and clots. It was concluded that human RBC stored at 4 degrees C for 75 days with SOD solution, recovery of levels of RBC-Hb and plasma-Hb were accorded with the requirements of "Basic Demands of Blood Station" in China.