Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.     

Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system

Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically aminoacylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.     

Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination

The effects of thermal treatment on Limulus amebocyte lysate (LAL) reagent were studied. Thermal resistances of enzymes and coagulogen in LAL reagent were evaluated by aggregometry and SDS-PAGE. Although enzyme activities of LAL reagent were completely lost after heating at temperatures above 60 °C for 10 min, gelating activities of coagulogen were retained even over 80 °C. Phenylmethanesulfonyl fluoride (PMSF; 1 mmol/mL), a strong non-specific serine-protease inhibitor, did not completely inactivate serine-protease activities of LAL. As a result, complete hydrolysis of coagulogen to coagulin was unexpectedly obtained. Solvent treatment of LAL was similar in effect to thermal treatment of LAL, but there were 2 problems: complete removal of solvent from samples and increased solution turbidity during preparation. To study the application of thermal-treated LAL, we conjugated it with titania particles. LAL-conjugated titania particles were obtained as small aggregates between titania nanoparticles and thermal-treated LAL (LAL-conjugated microbeads; LCM). When the mixture of LCMs and fresh LAL reagent was reacted with endotoxin an acute aggregation of LCMs was induced prior to the aggregate formation of LAL as monitored by stirring turbidimetry. This method, endotoxin microbeads aggregometry (EMA) may provide a rapid and sensitive method for endotoxin determination.     

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.     

Human Platelet Lysate for Good Manufacturing Practice-Compliant Cell Production

Numerous cell-based therapeutics are currently being tested in clinical trials. Human platelet lysate (HPL) is a valuable alternative to fetal bovine serum as a cell culture medium supplement for a variety of different cell types. HPL as a raw material permits animal serum-free cell propagation with highly efficient stimulation of cell proliferation, enabling humanized manufacturing of cell therapeutics within a reasonable timeframe. Providers of HPL have to consider dedicated quality issues regarding identity, purity, potency, traceability and safety. Release criteria have to be defined, characterizing the suitability of HPL batches for the support of a specific cell culture. Fresh or expired platelet concentrates from healthy blood donors are the starting material for HPL preparation, according to regulatory requirements. Pooling of individual platelet lysate units into one HPL batch can balance donor variation with regard to essential platelet-derived growth factors and cytokines. The increasingly applied pathogen reduction technologies will further increase HPL safety. In this review article, aspects and regulatory requirements of whole blood donation and details of human platelet lysate manufacturing are presented. International guidelines for raw materials are discussed, and defined quality controls, as well as release criteria for safe and GMP-compliant HPL production, are summarized.     

用细菌内毒素检查法检测巴氏灭活前后内毒素的变化

低温乙醇法生产白蛋白的过程中，通过对60℃、10h加温灭活病毒前后细菌内毒素含量的测定，证明巴氏灭活不仅能灭活病毒，还可以去除部分热原质。     

Magnetic Nanoparticles for Plasmid DNA Purification through Hydrophobic Interaction Chromatography

Magnetic poly(2-hydroxyethyl methacrylate-co-N-methacryloyl–L-phenyl alanine), p(HEMA-co-MAPA) nanoparticles were produced by emulsion polymerization technique. The nanoparticles were characterized by zetasizer, atomic force microscopy (AFM), transmission electron microscopy (TEM), electron spin resonance spectroscopy (ESR), and Fourier transformed infrared spectroscopy (FTIR) techniques. Experimental conditions were optimized for DNA adsorption in aqueous media. The effects of several factors (i.e., temperature, pH, equilibrium concentration of DNA, salt type, and ionic strength) on adsorption capacity were examined in batch studies. The maximum DNA adsorption capacity was 211.3 mg/g for nanoparticles consisting of magnetite. The adsorption isotherms and kinetics were also examined. The Langmuir model and pseudo second-order kinetics are the best fitted to the data. Following the ten adsorption-desorption cycles, the decrease in adsorption capacity was only 12%. Finally, plasmid DNA was purified from E.coli lysate by magnetite nanoparticles consisting of MAPA. The purity and molecular mass of purified plasmid DNA were determined by the use of agarose gel electrophoresis resulting in 7000 base pairs for molecular mass and OC pDNA form following elution.     

头孢孟多酯钠细菌内毒素检查法可行性研究

建立了头孢孟多酯钠细菌内毒素检查方法。按2005年版中国药典第2部收载的细菌内毒素检查法规定,用2个不同厂家的鲎试剂对其分别进行了检查。结果表明,采用0.5 EU/mL的鲎试剂并将样品质量浓度稀释至3.33 mg/mL时,对该实验无干扰作用,细菌内毒素限值为0.15 EU/mg。头孢孟多酯钠可用细菌内毒素检查法代替热原检查法进行检验,方法灵敏、可靠、方便、经济。     

Adjuvant‐Loaded Subcellular Vesicles Derived From Disrupted Cancer Cells for Cancer Vaccination

Targeted subunit vaccines for cancer immunotherapy do not capture tumor antigenic complexity, and approaches employing tumor lysate are often limited by inefficient antigen uptake and presentation, and low immunogenicity. Here, whole cancer cells are processed to generate antigen‐rich, membrane‐enclosed subcellular particles, termed “reduced cancer cells”, that reflect the diversity and breadth of the parent cancer cell antigen repertoire, and can be loaded with disparate adjuvant payloads. These vesicular particles enhance the uptake of the adjuvant payload, and potentiate the activation of primary dendritic cells in vitro. Similarly, reduced cancer cell‐associated antigens are more efficiently presented by primary dendritic cells in vitro than their soluble counterparts or lysate control. In mice, vaccination using adjuvant‐loaded reduced cancer cells facilitates the induction of antigen‐specific cellular and humoral immune responses. Taken together, these observations demonstrate that adjuvant‐loaded reduced cancer cells could be utilized in cancer vaccines as an alternative to lysate.     

Carbon nanotubes conjugated to tumor lysate protein enhance the efficacy of an antitumor immunotherapy

The biomedical applications of carbon nanotubes (CNTs) have attracted deep interest in recent years. Antitumor immunotherapy has the potential to improve the prognosis of cancer treatment but the efficacy of current immunotherapy generally needs further improvement. Multi-walled CNTs conjugated to tumor lysate protein are investigated as to whether they would enhance the efficacy of an immunotherapy employing a tumor-cell vaccine in a mouse model bearing the H22 liver cancer. The tumor cure rate is found to be markedly improved by CNTs conjugated to tumor lysate protein. The cellular antitumor immune reaction is also enhanced. Moreover, the observed antitumor immune response is relatively specific against the tumor intended for treatment. These findings suggest that CNTs may have a prospective role in the development of new antitumor immunotherapies.