Alternative Internal Standard Calibration of an Indirect Enzymatic Analytical Method for 2-MCPD Fatty Acid Esters.

An indirect enzymatic analysis method for the quantification of fatty acid esters of 2-/3-monochloro-1,2-propanediol (2/3-MCPD) and glycidol was developed, using the deuterated internal standard of each free-form component. A statistical method for calibration and quantification of 2-MCPD-d5, which is difficult to obtain, is substituted by 3-MCPD-d5 used for calculation of 3-MCPD. Using data from a previous collaborative study, the current method for the determination of 2-MCPD content using 2-MCPD-d5 was compared to three alternative new methods using 3-MCPD-d5. The regression analysis showed that the alternative methods were unbiased compared to the current method. The relative standard deviation (RSDR) among the testing laboratories was ≤ 15% and the Horwitz ratio was ≤ 1.0, a satisfactory value.

Collaborative Study of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils.

A collaborative study was conducted to evaluate an indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. The method is characterized by the use of Candida rugosa lipase, which hydrolyzes the esters at room temperature in 30 min. Hydrolysis and bromination steps convert esters of 3-MCPD, 2-MCPD, and glycidol to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol, respectively, which are then derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometry. In a collaborative study involving 13 laboratories, liquid palm, solid palm, rapeseed, and rice bran oils spiked with 0.5-4.4 mg/kg of esters of 3-MCPD, 2-MCPD, and Gly were analyzed in duplicate. The repeatability (RSDr) were < 5% for five liquid oil samples and 8% for a solid oil sample. The reproducibility (RSDR) ranged from 5% to 18% for all oil samples. These RSDR values were considered satisfactory because the Horwitz ratios were ≤ 1.3% for all three analytes in all oil samples. This method is applicable to the quantification of 3-MCPD, 2-MCPD, and Gly esters in edible oils.

Optimization of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils.

We developed a novel, indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. Using this method, the ester analytes were rapidly cleavaged by Candida rugosa lipase at room temperature for 0.5 h. As a result of the simultaneous hydrolysis and bromination steps, 3-MCPD esters, 2-MCPD esters, and glycidyl esters were converted to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol (3-MBPD), respectively. After the addition of internal standards, the mixtures were washed with hexane, derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometer (GC-MS). The analytical method was evaluated in preliminary and feasibility studies performed by 13 laboratories. The preliminary study from 4 laboratories showed the reproducibility (RSD R ) of < 10% and recoveries in the range of 102-111% for the spiked 3-MCPD and 2-MCPD in extra virgin olive (EVO) oil, semi-solid palm oil, and solid palm oil. However, the RSDR and recoveries of Gly in the palm oil samples were not satisfactory. The Gly content of refrigerated palm oil samples decreased whereas the samples at room temperature were stable for three months, and this may be due to the depletion of Gly during cold storage. The feasibility studies performed by all 13 laboratories were conducted based on modifications of the shaking conditions for ester cleavage, the conditions of Gly bromination, and the removal of gel formed by residual lipase. Satisfactory RSDR were obtained for EVO oil samples spiked with standard esters (4.4% for 3-MCPD, 11.2% for 2-MCPD, and 6.6% for Gly).

Identification and characterisation of structural maintenance of chromosome 1 (smc1) mutants of Coprinopsis cinerea.

A Coprinopsis cinerea homokaryotic fruiting strain was mutagenised, identifying a mutant that exhibited a hyphal growth temperature sensitive defect and hyphal knot development defect at an early fruiting stage, even at the hyphal growth permissive temperature. Microscopic observation suggested that the mutant nuclei exhibited defects in the metaphase to anaphase transition at the restrictive temperature. The gene in which the mutation occurred was cloned, sequenced and determined to be homologous to smc1. Sequence analyses of the mutant revealed deletion of 28 base pairs in the 19th intron of the Cc.smc1 gene, resulting in complete failure of splicing of that intron and in insertion of 14 amino acids in the C-terminal region of the Cc.Smc1 protein. We isolated eight hyphal growth revertants and identified four intragenic suppressors. All were the result of amino acid substitutions in the C-terminal region. Three of the suppressors caused reversion of the arrest in an early fruiting stage. One of the suppressors exhibited cold sensitivity and failed to suppress the fruiting defect, suggesting that flexibility of a lobe in the C-terminal region is important for proper function of Cc.Smc1.

Passive soma facilitates submillisecond coincidence detection in the owl's auditory system.

Neurons of the avian nucleus laminaris (NL) compute the interaural time difference (ITD) by detecting coincident arrivals of binaural signals with submillisecond accuracy. The cellular mechanisms for this temporal precision have long been studied theoretically and experimentally. The myelinated axon initial segment in the owl's NL neuron and small somatic spikes observed in auditory coincidence detector neurons of various animals suggest that spikes in the NL neuron are generated at the first node of Ranvier and that the soma passively receives back-propagating spikes. To investigate the significance of the "passive soma" structure, we constructed a two-compartment NL neuron model, consisting of a cell body and a first node, and systematically changed the excitability of each compartment. Here, we show that a neuron with a less active soma achieves higher ITD sensitivity and higher noise tolerance with lower energy costs. We also investigate the biophysical mechanism of the computational advantage of the "passive soma" structure by performing sub- and suprathreshold analyses. Setting a spike initiation site with high sodium conductance, not in the large soma but in the small node, serves to amplify high-frequency input signals and to reduce the impact and the energy cost of spike generation. Our results indicate that the owl's NL neuron uses a "passive soma" design for computational and metabolic reasons.