Ku70 Is Required for Late B Cell Development and Immunoglobulin Heavy Chain Class Switching

Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70^(−/−) (K70T) mice, like recombination activating gene (RAG)-1– or RAG-2–deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653–665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921–929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig λ light chain transgene (referred to as K70T/HL mice). Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J. Manis, E. Sonoda, K. Rajewsky, and F. Alt. 1998. Int. Immunol. 10:325–332). K70T/HL mice had significant numbers of peripheral surface IgM^+ B cells, which generated serum IgM levels similar to those of R2T/HL mice. However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes. In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells. However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h. We conclude that Ku70 is required for the generation of B cells that have undergone Ig HC class switching. Potential roles for Ku70 in the CSR process are discusse

Defective DNA Strand Break Repair Causes Chromosomal Instability and Accelerates Liver Carcinogenesis in Mice

Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end-joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ-deficient (Ku70-/-), heterozygote (Ku70 +/-), and wild-type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70-/- mice injected with DEN displayed accelerated HCC development. Ku70-/- HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70-/- HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. Conclusion: These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC

Estimation of Fracture Size and Probability Density Function by Setting Scanlines in Rectangular Sampling Window

Rock masses are very important materials in geotechnical engineering. In engineering rock mass, fracture is the relatively weak part of mechanical strength in rock mass and is the most important factor controlling the deformation, damage, and permeability of rock mass. Therefore, investigating fractures is very important for characterizing rock mass. This paper proposed a new approach by using uniformly equidistant orthogonal scanlines. Within the study context, the solution formula of fracture size is derived by establishing the space intersection model of arbitrary fracture and scanline, rectangular window, and a rectangular box with a rectangular window. Then, fractures were randomly generated in a certain size cube and compared with the traditional Kulatilake trace length integral evaluation method. The study results have shown that the proposed method is more reasonable and accurate. Then, this method was applied to an adit of Songta Hydropower Station. Finally, a new fracture diameter probability density estimation method was proposed, the fracture diameter of the normal distribution was verified, and the parameters of the probability density function obtained by the scanlines method were in agreement with the initial set parameters. In summary, the proposed scanlines method can well estimate the mean value of the fracture diameter and the probability density function of the fracture size

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Hyperproduction of Alcohol Using Yeast Fermentation in Highly Concentrated Molasses Medium

Cane molasses, a major byproduct in the sugar industry, is generally consumed for alcohol production. However, the alcohol production process needs to overcome three major challenges including increasing the productivity of alcohol fermentation, lowering the energy consumption for alcohol conversion and decreasing the environmental pollution caused by the alcoholic yeast fermentation process. To meet these challenges, a screening process was conducted using 13 high osmotic tolerant yeast strains. Among the strains, a Saccharomyces cerevisiae   strain 1912 was found to produce high alcohol concentrations during fermentation with high starting molasses concentrations such as 50% (W/V) molasses. In the test, 13.6% (V/V) alcohol was produced in the molasses fermentation broth after 72 h of incubation with an initial Yunnan molasses concentration of 50% in a 5 L fermentor. 15.0% (V/V) alcohol was obtained after 48 h of fermentation in shaking flasks containing 30% (W/V) initial total sugar concentration in diluted molasses. The performance of this strain in the shaking flasks was successfully scaled up to a 5-L fermentor vessel. Strain 1912 seems to be a better alcohol producer than the currently used alcohol production strain 2.1190

Target Detection over the Diurnal Cycle Using a Multispectral Infrared Sensor

When detecting a target over the diurnal cycle, a conventional infrared thermal sensor might lose the target due to the thermal crossover, which could happen at any time throughout the day when the infrared image contrast between target and background in a scene is indistinguishable due to the temperature variation. In this paper, the benefits of using a multispectral-based infrared sensor over the diurnal cycle have been shown. Firstly, a brief theoretical analysis on how the thermal crossover influences a conventional thermal sensor, within the conditions where the thermal crossover would happen and why the mid-infrared (3~5 μm) multispectral technology is effective, is presented. Furthermore, the effectiveness of this technology is also described and we describe how the prototype design and multispectral technology is employed to help solve the thermal crossover detection problem. Thirdly, several targets are set up outside and imaged in the field experiment over a 24-h period. The experimental results show that the multispectral infrared imaging system can enhance the contrast of the detected images and effectively solve the failure of the conventional infrared sensor during the diurnal cycle, which is of great significance for infrared surveillance applications