Activation of STAT3 stimulates AHSP expression in K562 cells

Studies on the chaperone proteinα-hemoglobin stabilizing protein(AHSP)reveal that abundant AHSP in erythroid cells enhance the cells’tolerance to oxidative stress imposed by excessα-hemoglobin in pathological conditions.However,the potential intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown.The present study examined the effect and molecular mechanism of STAT3,an oxidative regulator,on the expression of AHSP.AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells.Regulation of AHSP in oxidative circumstance was then examined inα-globin-overloaded K562 cells,and real-time PCR showed strengthened expression of both AHSP and STAT3.ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and uponα-globin overexpression.Dual luciferase reporter assays of the wildtype and mutated SB3 element,an IL-6RE site,in the AHSP promoter in K562 cells highlighted the direct regulatory effect of STAT3 on AHSP gene.Finally,direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA assays.Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway,and provides clues to the therapeutic strategy for AHSP enhancement.     

The Imaging of Insulinomas Using a Radionuclide-Labelled Molecule of the GLP-1 Analogue Liraglutide: A New Application of Liraglutide

Objective

This study explores a new, non-invasive imaging method for the specific diagnosis of insulinoma by providing an initial investigation of the use of ¹²⁵I-labelled molecules of the glucagon-like peptide-1 (GLP-1) analogue liraglutide for in vivo and in vitro small-animal SPECT/CT (single-photon emission computed tomography/computed tomography) imaging of insulinomas.

Methods

Liraglutide was labelled with ¹²⁵I by the Iodogen method. The labelled ¹²⁵I-liraglutide compound and insulinoma cells from the INS-1 cell line were then used for in vitro saturation and competitive binding experiments. In addition, in a nude mouse model, the use of ¹²⁵I-liraglutide for the in vivo small-animal SPECT/CT imaging of insulinomas and the resulting distribution of radioactivity across various organs were examined.

Results

The labelling of liraglutide with ¹²⁵I was successful, yielding a labelling rate of approximately 95% and a radiochemical purity of greater than 95%. For the binding between ¹²⁵I-liraglutide and the GLP-1 receptor on the surface of INS-1 cells, the equilibrium dissociation constant (K_d) was 128.8±30.4 nmol/L(N = 3), and the half-inhibition concentration (IC₅₀) was 542.4±187.5 nmol/L(N = 3). Small-animal SPECT/CT imaging with ¹²⁵I-liraglutide indicated that the tumour imaging was clearest at 90 min after the ¹²⁵I-liraglutide treatment. An examination of the in vivo distribution of radioactivity revealed that at 90 min after the ¹²⁵I-liraglutide treatment, the target/non-target (T/NT) ratio for tumour and muscle tissue was 4.83±1.30(N = 3). Our study suggested that ¹²⁵I-liraglutide was predominantly metabolised and cleared by the liver and kidneys.

Conclusion

The radionuclide ¹²⁵I-liraglutide can be utilised for the specific imaging of insulinomas, representing a new non-invasive approach for the in vivo diagnosis of insulinomas.     

Influence of Al3+ on the titer of spiramycin and effective components in fermentor

Spiramycin is a multicomponent antibiotic, and different components have different antibacterial activities. In Streptomyces spiramyceticus 16-10-2, spiramycin II and spiramycin III (SPMII and SPMIII) are the main components, while spiramycin I (SPMI) needs to be controlled below 12%. Based on this, the influences of Al³⁺ on total spiramycin titer and components were investigated in this work. Those experiments were mainly performed in 15?L fermentor and Al³⁺ made a great improvement in spiramycin titer. The optimal adding concentration and adding time of Al³⁺ were 0.32?g/L at 12?hr. Under this condition, spiramycin titer was increased by 19.51% compared with the control. Moreover, the percentage of SPMII and SPMIII was increased by 7.14%. At the same time, the time of mycelia autolysis was lengthened. In addition, the specific activities of acetyl-CoA synthetase, acetate kinase, acetylphosphotransferase, and acylating enzyme were much higher than those of control. The content of acetic acid and succinic acid was beyond 3 and 4.5 times than that of control, respectively.     

Comprehensive insights into the influence of supplemental green light on the photosynthesis of ginger (Zingiber officinale Roscoe)

Although green light is not considered to contribute to the photosynthesis of plants, the photosynthesis of ginger, a dual-purpose vegetable used as a medicine and food, is affected by the green wave band. In this study, the supplementary green band of sunlight (SG) increased the net photosynthetic rate (Pn), maximal photochemical efficiency of PSII (Fv/Fm), and actual photochemical efficiency of PSII (Y(II)) compared with the sunlight treatment (S). The Pn and Fv/Fm of the SG treatment were higher than those of the white light (W) treatment, while the Pn and Fv/Fm of the green light (G) treatment alone were lower than those of the W treatment. Further analysis found that the minimal fluorescence (Fo) of the S treatment increased, especially at noon, while the Fo of the SG treatment decreased. Similarly, the Fo of the W treatment increased significantly, while the Fo of the white–green mixed light (WG) treatment decreased. The relative fluorescence values of the K-J-I bands in the SG and WG treatments were lower than those in the S and W treatments, respectively. The photochemical quenching (qP) of the WG treatment was higher than that of the W treatment, while the primary thermal losses corresponded to the sum of nonregulated heat dissipation and fluorescence emission (Y(NO)) of the WG treatment was lower than that of the W treatment. The SG treatment reduced the accumulation of plastoglobules but increased the accumulation of starch granules and leaf thickness. Moreover, the green band supplemented with white light significantly increased the biomass of the aboveground plant parts and promoted the active growth of the aboveground parts. Supplementing green light plays a regulatory role in ginger based on the following four points. First, it effectively promotes the transfer of electrons between the acceptor side of photosystem II; second, it optimizes ginger photosynthesis; third, it alleviates strong light stress by reducing the accumulation of reactive oxygen species; and fourth, it promotes heat dissipation and reduces the rapid burst of active oxygen in the chloroplast caused by excess energy. In summary, green light can significantly optimize the photosynthetic characteristics of ginger.

     

The role of microvesicles and its active molecules in regulating cellular biology

Cell‐derived microvesicles are membrane vesicles produced by the outward budding of the plasma membrane and released by almost all types of cells. These have been considered as another mechanism of intercellular communication, because they carry active molecules, such as proteins, lipids and nucleic acids. Furthermore, these are present in circulating fluids, such as blood and urine, and are closely correlated to the progression of pathophysiological conditions in many diseases. Recent studies have revealed that microvesicles have a dual effect of damage and protection of receptor cells. However, the nature of the active molecules involved in this effect remains unclear. The present study mainly emphasized the mechanism of microvesicles and the active molecules mediating the different biological effects of receptor cells by affecting autophagy, apoptosis and inflammation pathways. The effective ways of blocking microvesicles and its active molecules in mediating cell damage when microvesicles exert harmful effects were also discussed.     

VKORC1 rs2359612C allele is associated with increased risk of coronary artery disease in the presence of coronary calcification

VKORC1 genetic polymorphisms affect warfarin dose response, aortic calcification, and the susceptibility of coronary artery disease as shown in our previous study. Little is known regarding the association of VKORC1 polymorphisms with coronary artery calcification (CAC) and the role of CAC in the association with coronary artery disease (CAD). Due to a natural haplotype block in the VKORC1 gene in Chinese, polymorphism rs2359612 was analyzed in a case–control study and a prospective study. The case–control study included 464 CAD patients with non-calcified plaque (NCP), 562 CAD patients with mixed calcified plaque (MCP), 492 subjects with calcified plaque (CP), and 521 controls. The rs2359612C was only associated with increased risk of MCP, the CAD in the presence of CAC; the odds ratio was 1.397 (95 % CI 1.008–1.937, P < 0.05), which was replicated in the second independent population. On the contrary, a negative correlation was observed between rs2359612 and log-transformed Agatston score, and rs2359612 was negatively associated with the number of calcified vessels. Moreover, in a prospective study including 849 CAD patients undergoing revascularization, rs2359612C predicted a higher incidence of cardiovascular events in MCP subgroup; the relative risk was 1.435 (95 % CI 1.008–2.041, P = 0.045), which was not observed in the NCP subgroup. We conclude that the rs2359612C was associated with a higher risk of CAD in the presence of CAC and a higher incidence of cardiovascular events in CAD patients with CAC, but a lower coronary calcification. VKORC1 polymorphisms may be associated with the endophenotype of CAD, calcification-related atherosclerosis.     

Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model

Objective

Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.

Methods

Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO₂ cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.

Results

Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.

Conclusions

These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.     

Structural and Functional Insights into Saccharomyces cerevisiae Riboflavin Biosynthesis Reductase RIB7

Saccharomyces cerevisiae RIB7 (ScRIB7) is a potent target for anti-fungal agents because of its involvement in the riboflavin biosynthesis pathway as a NADPH-dependent reductase. However, the catalytic mechanism of riboflavin biosynthesis reductase (RBSRs) is controversial, and enzyme structure information is still lacking in eukaryotes. Here we report the crystal structure of Saccharomyces cerevisiae RIB7 at 2.10 Å resolution and its complex with NADPH at 2.35 Å resolution. ScRIB7 exists as a stable homodimer, and each subunit consists of nine central β-sheets flanked by five helices, resembling the structure of RIB7 homologues. A conserved G⁷⁶-X-G⁷⁸-X_n-G¹⁸¹-G¹⁸² motif is present at the NADPH pyrophosphate group binding site. Activity assays confirmed the necessity of Thr79, Asp83, Glu180 and Gly182 for the activity of ScRIB7. Substrate preference of ScRIB7 was altered by mutating one residue (Thr35) to a Lysine, implying that ScRIB7 Thr35 and its corresponding residue, a lysine in bacteria, are important in substrate-specific recognition.     

Specificity of miR-378a-5p targeting rodent fibronectin

One criterion for microRNA identification is based on their conservation across species, and prediction of miRNA targets by empirical approaches using computational analysis relies on the presence of conservative mRNA 3′UTR. Because most miRNA target sites identified are highly conserved across different species, it is not clear whether miRNA targeting is species-specific. To predict miRNA targeting, we aligned all available fibronectin 3′UTRs and observed significant conservation of all 20 species. Twelve miRNAs were predicted to target most fibronectin 3′UTRs, but rodent fibronectin showed potential binding sites specific for five different miRNAs. One of them, the miR-378a-5p, contained a complete matching seed-region for all rodent fibronectin, which could not be found in any other species. We designed experiments to test whether the species-specific targeting possessed biological function and found that expression of miR-378a-5p decreased cancer cell proliferation, migration, and invasion, resulting in inhibition of tumor growth. Silencing fibronectin expression produced similar effects as miR-378a-5p, while transfection with a construct targeting miR-378-5p produced opposite results. Tumor formation assay showed that enhanced expression of fibronectin in the stromal tissues as a background environment suppressed tumor growth, while increased fibronectin expression inside the tumor cells promoted tumor growth. This was likely due to the different signaling direction, either inside-out or outside-in signal. Our results demonstrated that species-specific targeting by miRNA could also exert functional effects. Thus, one layer of regulation has been added to the complex network of miRNA signaling.