To identify genetic susceptibility loci for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in the Chinese population, we carried out a genome-wide association study (GWAS) in 2,514 chronic HBV carriers (1,161 HCC cases and 1,353 controls) followed by a 2-stage validation among 6 independent populations of chronic HBV carriers (4,319 cases and 4,966 controls). The joint analyses showed that HCC risk was significantly associated with two independent loci: rs7574865 at STAT4, Pmeta = 2.48 × 10−10, odds ratio (OR) = 1.21; and rs9275319 at HLA-DQ, Pmeta = 2.72 × 10−17, OR = 1.49. The risk allele G at rs7574865 was significantly associated with lower mRNA levels of STAT4 in both the HCC tissues and nontumor tissues of 155 individuals with HBV-related HCC (Ptrend = 0.0008 and 0.0002, respectively). We also found significantly lower mRNA expression of STAT4 in HCC tumor tissues compared with paired adjacent nontumor tissues (P = 2.33 × 10−14).

An improved strategy was developed for the high-density culture of Magnetospirillum gryphiswaldense strain MSR-1 and large-scale magnetosome production in both 7.5-and 42-liter autofermentors. By using a nutrientbalanced feeding strategy and the replacement of carbon and nitrogen sources to reduce accumulation of Na ؉ and Cl ؊ ions, we reduced the factors that tend to inhibit cell growth, particularly the increase of osmotic potential. Semicontinuous culture was thereby achieved in the autofermentor for the first time. When the cells were harvested at 36 and 73 h, magnetosome yields (dry weight) as high as 168.3 and 83.5 mg/liter/day, respectively, were achieved. These values were, respectively, approximately 10 and 5 times higher than the yields achieved in previous studies and represent a significant improvement in magnetosome production efficiency.Biomineralized magnetosomes (chains of magnetite crystals found in prokaryotes) have attracted commercial interest because of their narrow size range, good dispersibility, and biomembrane enclosure. Previous studies have addressed a variety of applications and properties, including enzyme immobilization (5), gene delivery system (16), cell separation (19), drug carriers (11, 12), immunoassays (4, 14), protein and multisubunit enzyme complexes (7,20), and use of microorganisms per se for mineral recovery (13). Because of the highly restrictive culture conditions for magnetotactic bacteria, in terms of the dissolved oxygen concentration (dO 2 ) (3, 17), nutrients, etc., the yields of both magnetosomes and their host microorganisms under artificial culture tend to be low (10, 18). A long-standing research goal of our laboratory is improved large-scale production of cells and magnetosomes.In a previous study using fed-batch culture techniques (10), we achieved maximal cell density (optical density at 565 nm [OD 565 ] of 7.24, cell dry weight of 2.17 g/liter [0.87 g/liter/day], and magnetosome dry weight of 41.7 mg/liter [16.7 mg/liter/ day]). Through further optimization of culture temperature, pH, dO 2 , and nutrients, we achieved an OD 565 value of 12 in a 7.5-liter fermentor after 40 h of culture (unpublished data). In revising our previous feeding strategy, we focused on supplementation of carbon and nitrogen sources but ignored two possible factors that could inhibit cell growth: (i) nutrient limitation arising during fermentation process and (ii) the accumulation of Na ϩ and Cl Ϫ in a fermentor fed with sodium lactate and ammonium chloride. By replacing the carbon and nitrogen sources and using an optimally nutrient-balanced feeding strategy, in a 7.5-liter fermentor after 44 h, we achieved an OD 565 of 30.4, a cell dry weight of 7.59 g/liter (3.8 g/liter/ day), and a magnetosome dry weight of 225.53 mg/liter (112.77 mg/liter/day). In a larger (42-liter) fermentor, after 44 h, we achieved an OD 565 of 42, a cell dry weight of 9.16 g/liter (4.58 g/liter/day), and a magnetosome dry weight of 356.52 mg/liter (178.26 mg/liter/day). The efficiency of magnetosome produ...

BackgroundMagnetotactic bacteria have long intrigued researchers because they synthesize intracellular nano-scale (40-100 nm) magnetic particles composed of Fe3O4, termed magnetosomes. Current research focuses on the molecular mechanisms of bacterial magnetosome formation and its practical applications in biotechnology and medicine. Practical applications of magnetosomes are based on their ferrimagnetism, nanoscale size, narrow size distribution, dispersal ability, and membrane-bound structure. However, the applications of magnetosomes have not yet been developed commercially, mainly because magnetotactic bacteria are difficult to cultivate and consistent, high yields of magnetosomes have not yet been achieved.ResultsWe report a chemostat culture technique based on pH-stat feeding that yields a high cell density of Magnetospirillum gryphiswaldense strain MSR-1 in an auto-fermentor. In a large-scale fermentor, the magnetosome yield was significantly increased by adjusting the stirring rate and airflow which regulates the level of dissolved oxygen (DO). Low concentration of sodium lactate (2.3 mmol l-1) in the culture medium resulted in more rapid cell growth and higher magnetosome yield than high concentration of lactate (20 mmol l-1). The optical density of M. gryphiswaldense cells reached 12 OD565 nm after 36 hr culture in a 42 L fermentor. Magnetosome yield and productivity were 83.23 ± 5.36 mg l-1 (dry weight) and 55.49 mg l-1 day-1, respectively, which were 1.99 and 3.32 times higher than the corresponding values in our previous study.ConclusionsCompared to previously reported methods, our culture technique with the MSR-1 strain significantly increased cell density, cell yield, and magnetosome yield in a shorter time window and thus reduced the cost of production. The cell density and magnetosome yield reported here are the highest so far achieved with a magnetotactic bacteria. Refinement of this technique will enable further increase of cell density and magnetosome yield.

Magnetotactic bacteria (MTB) synthesize unique organelles termed "magnetosomes," which are membraneenclosed structures containing crystals of magnetite or greigite. Magnetosomes form a chain around MamK cytoskeletal filaments and provide the basis for the ability of MTB to navigate along geomagnetic field lines in order to find optimal microaerobic habitats. Genomes of species of the MTB genus Magnetospirillum, in addition to a gene encoding the tubulin-like FtsZ protein (involved in cell division), contain a second gene termed "ftsZ-like," whose function is unknown. In the present study, we found that the ftsZ-like gene of Magnetospirillum gryphiswaldense strain MSR-1 belongs to a 4.9-kb mamXY polycistronic transcription unit. We then purified the recombinant FtsZ-like protein to homogeneity. The FtsZ-like protein efficiently hydrolyzed ATP and GTP, with ATPase and GTPase activity levels of 2.17 and 5.56 mol phosphorus per mol protein per min, respectively. The FtsZ-like protein underwent GTP-dependent polymerization into long filamentous bundles in vitro. To determine the role of the ftsZ-like gene, we constructed a ftsZ-like mutant (⌬ftsZ-like mutant) and its complementation strain (⌬ftsZ-like_C strain). Growth of ⌬ftsZ-like cells was similar to that of the wild type, indicating that the ⌬ftsZ-like gene is not involved in cell division. Transmission electron microscopic observations indicated that the ⌬ftsZ-like cells, in comparison to wild-type cells, produced smaller magnetosomes, with poorly defined morphology and irregular alignment, including large gaps. Magnetic analyses showed that ⌬ftsZ-like produced mainly superparamagnetic (SP) magnetite particles, whereas wildtype and ⌬ftsZ-like_C cells produced mainly single-domain (SD) particles. Our findings suggest that the FtsZ-like protein is required for synthesis of SD particles and magnetosomes in M. gryphiswaldense.Magnetotactic bacteria (MTB) can orient themselves along geomagnetic field lines and search for microaerophilic environments. These capabilities are based on unique prokaryotic organelles termed magnetosomes (3). Magnetosomes are nanometer-size magnetic particles of iron oxide (magnetite; Fe 3 O 4 ) or iron sulfide (greigite; Fe 3 S 4 ) (4, 5, 45), enclosed within intracytoplasmic vesicles of the magnetosome membrane (MM) (3, 43). Magnetosome formation is a complex process involving vesicle formation, iron transportation, nucleation and growth of magnetite crystals, and their assembly into chain-like structures. A model for magnetosome formation has been proposed by Komeili (18) and Schüler (44). According to this model, magnetosome vesicles are invaginated from the inner membrane, and protein sorting to the MM occurs concurrently. The protein MamA was suggested to activate magnetosome vesicles for magnetite biomineralization (19). With the help of the MamK and MamJ proteins, the membrane invaginations are then assembled into a chain structure. The bacterial actin-like MamK can form filaments required for maintaining magnetosome organization a...