Tailoring Oxidation Responsiveness of Gold Nanoclusters via Ligand Engineering for Imaging Acute Kidney Injury.

Gold nanoclusters (AuNCs) have shown great promise for in vivo imaging because of their definable structure, tunable photoluminescence (PL), and desired renal clearance. However, current understanding of the responsiveness of AuNCs to biological substances is still limited, which may hamper their biomedical applications. Herein, we explore the oxidation responsiveness of near-infrared II (NIR-II) luminescent AuNCs capped with two different ligands, which can be optimized for high-efficiency NIR-II PL imaging of mice acute kidney injury (AKI) featuring high-level peroxynitrite anions (ONOO-). We found that in the presence of ONOO-, N-acetylcysteine-capped AuNCs (NAC-AuNCs) tended to be oxidized more easily than that capped with the macromolecular mercapto-ß-cyclodextrin (CDS-AuNCs), resulting in the aggregation of NAC-AuNCs into large-sized assemblies, which was not observed in CDS-AuNCs. The oxidation-triggered morphology, composition, and NIR-II PL changes in NAC-AuNCs were then systematically studied. We finally demonstrated that NAC-AuNCs can be implemented for sensitive NIR-II PL imaging of mice AKI, facilitated by the synergetic in situ AuNC aggregation and decreased glomerular filtration rate (GFR) in the injured kidney, which outperforms the methods solely based on the decreased GFR effect. Therefore, this work highlights the critical significance of ligand engineering in AuNCs and may motivate future design of AuNCs for diverse bioimaging applications.

Enzyme kinetics and molecular modeling studies of G6PD(Mahidol) associated with acute hemolytic anemia.

G6PD(Mahidol) enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PD(Mahidol) enzyme was characterized by molecular modeling to understand its kinetics. G6PD(Mahidol), G6PD(G487A) and G6PD(WT) proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PD(WT), the Km, and Vmax of NADP+ with G6PD(G487A) were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PD(G487A) showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PD(G487A) was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PD(G487A) alpha-helix d and beta-strand E, besides the conformation of beta-strand D. In conclusion, the biochemical and structural properties of G6PD(G487A) and G6PD(WT) enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.

Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of Southwestern China.

The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2-13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen independent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.

A new G6PD knockdown tumor-cell line with reduced proliferation and increased susceptibility to oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD) has been implicated in the regulation of cellular antioxidative mechanisms. Tumor cells often lose the balance of oxidation and antioxidation, but the role of G6PD in such an imbalance is still largely unknown. To investigate the related function of G6PD in tumor cells, we established a stable line of A375 human melanoma cells with G6PD gene knockdown by a shRNA lentiviral cloning and expression system. The A375-G6PDDelta cells displayed the stable GFP coexpression after repeated freeze-thaw cycles and multiple passages, accompanied by an 88.83% suppression of the endogenous G6PD expression and a 78.47% decrease in G6PD activity. In comparison with the A375-WT cells, they were characterized by a reduced proliferation with the MTT proliferation assay, a 25% decrease in colony-forming efficiency, and an up to 40% increase of apoptotic rate with flow cytometry analysis. When further challenged by diamide-induced oxidative stress, these cells showed that a median lethal dose (LD(50)) of 1.2 mM decreased from that of the A375-WT cells (1.8 mM), and levels of NADPH and GSH decreased by 2.4-, 8.8-fold, respectively, with a 7.3-fold increase of H(2)O(2), as those of A375-WT cells. These results demonstrated that A375-G6PDDelta is a new, stable G6PD-deficient human tumor cell line, and that silencing G6PD expression decreased tumor-cell proliferation and enhanced apoptosis. In addition, G6PD gene knockdown rendered tumor cells more susceptible to diamide-induced oxidative stress. Together, our data support the important functions of G6PD in the regulation of cell growth and antioxidative capacity of tumor cells.