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Jonathan Okamura’s riveting account of Myles Yutaka Fukunaga’s 1928 kidnapping and murder of ten-year-old George Gill Jamieson takes you back to interwar Hawai'i, a pivotal moment in the territory’...

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (

Gross, histological, and immunohistochemical changes in the combs of chickens after bile duct ligation (BDL) are described. Gross reductions in comb size and volume and lower serum testosterone levels were evident in chickens after BDL. Histologically, atrophic combs were characterized by reduced blood capillary diameter, decreased acid mucopolysaccharides, thinning of the stratum germinativum of the epidermis and dermis, and reduced immunostaining intensity of androgen receptors. These results suggest that the affected cells in atrophic combs are androgen targets. BDL caused testicular atrophy in chickens, a primary complication of liver disease, and the resultant low serum testosterone levels subsequently caused atrophy of the comb. In other words, the atrophy of the comb observed in BDL chickens was a secondary complication of liver dysfunction that simulated the effects of liver disease.

A method has been developed for the determination of diphenylarsinic acid (DPAA) and phenylarsonic acid (PAA) in water samples by high-performance liquid chromatography with inductively coupled plasma mass spectrometry. DPAA and PAA could be separated from five arsenic species {As(III), As(V), monomethylarsonic acid, dimethylarsinic acid and trimetylarsine oxide} by using a column packed with hydrophilic-resin-based cation-exchange resin and a gradient elution with nitric acid-ammonium nitrate. Good linearity (coefficient of correlation ≥ 0.998) of the calibration curve was obtained in the concentration range from 2 μg/l to 200 μg/l for DPAA and PAA. In addition, a solid-phase extraction method was developed to concentrate DPAA and PAA. The recoveries of DPAA and PAA from spiked groundwater samples were nearly 100% and the coefficients of variation were 2.6∼2.9 %(n = 5).

The temperature dependence of the negative nonlinear absorption (NNA) effect was investigated in an erbium-doped yttrium aluminum garnet (YAG) crystal using a 787.3 nm laser diode. The NNA effect was obtained in a temperature range of 15 to 400 K. An absorption peak corresponding to both the 4 I 15/2-4 I 9/2 and 4 I 13/2-2 H 11/2 transitions of Er3+ in the YAG at 400 K was displaced from that at 300 K. It is considered that the NNA effect weakened at high temperature because the absorption cross section decreased due to the shift of the absorption peak. In addition, the dependence of the NNA effect on the modulation degree was investigated. The characteristic of the increase in the incident laser intensity was almost the same as that for the decrease. It was confirmed that the mechanism of the NNA effect can be explained by calculating the rate equations based on an analytical model for a five-level system of the Er3+ ion. It is suggested that the NNA effect weakened at the low temperature because the number of phonons decreased due to the low temperature.