Is amino acid racemization a useful tool for screening for ancient DNA in bone?

Many rare and valuable ancient specimens now carry the scars of ancient DNA research, as questions of population genetics and phylogeography require larger sample sets. This fuels the demand for reliable techniques to screen for DNA preservation prior to destructive sampling. Only one such technique has been widely adopted: the extent of aspartic acid racemization (AAR). The kinetics of AAR are believed to be similar to the rate of DNA depurination and therefore a good measure of the likelihood of DNA survival. Moreover, AAR analysis is only minimally destructive. We report the first comprehensive test of AAR using 91 bone and teeth samples from temperate and high-latitude sites that were analysed for DNA. While the AAR range of all specimens was low (0.02–0.17), no correlation was found between the extent of AAR and DNA amplification success. Additional heating experiments and surveys of the literature indicated that d/l Asx is low in bones until almost all the collagen is lost. This is because aspartic acid is retained in the bone within the constrained environment of the collagen triple helix, where it cannot racemize for steric reasons. Only if the helix denatures to soluble gelatin can Asx racemize readily, but this soluble gelatine is readily lost in most burial environments. We conclude that Asx d/l is not a useful screening technique for ancient DNA from bone.     

False positive rate in newborn screening for congenital adrenal hyperplasia (CAH)-ether extraction reveals two distinct reasons for elevated 17α-hydroxyprogesterone (17-OHP) values

Background

While the sensitivity of newborn screening for the salt wasting form of congenital adrenal hyperplasia (CAH) is good, the positive predictive value is poor due to the high false positive rate of the immunological assays for 17-OHP. Cross-reactivity with steroid sulfates is one of the main causes for false positive results. Several approaches have been described to improve CAH screening: adjusting cut-off levels to gestational age or birth weight, and second-tier molecular genetic analysis or second-tier liquid chromatography-tandem mass spectrometry (TMS).

Methods

17-OHP was extracted with diethyl ether from dried blood spots in order to separate 17-OHP from polar steroids (like steroid sulfates). The dried ether extracts of calibrators, controls, and patient samples were redissolved and measured with the 17-OHP test kit (Wallac).

Results

760 normal, 1049 false positive, and 232 samples of confirmed cases with CAH were analysed. Mean 17-OHP values were significantly lower after extraction: Normal samples: 17.5 nmol/L vs. 3.2 nmol/L; false positive samples: 97.0 nmol/L vs. 25.9 nmol/L; CAH: 275 nmol/L vs. 205 nmol/L. With a cut-off value of 11.9 nmol/L (mean + 3 SD of the normal values), 404 of the false positives turned out to be normal. Ether extraction revealed two distinct subgroups of initially false positives rather than a continuum with normal distribution of 17-OHP values.

Conclusion

Diethyl ether extraction provided evidence for two causes of false positive results in CAH screening. It reduced the rate of false positives by about 40% without loss of sensitivity.     

高强度聚焦超声换能器温度场的数值仿真

相控阵高强度聚焦超声换能器可以通过换能器上不同阵元发射超声波的时间不同来实现变焦、多焦点。该论文应用Westervelt方程的近似式,结合Pennes热传导方程,以人体乳房为例,FDTD(finite difference time domain)仿真对比研究平面阵列相控聚焦换能器与曲面阵列相控聚焦换能器形成温度场的特性,同时数值仿真研究不同占空比的正弦激励函数、不同治疗频率、声强对曲面阵列相控聚焦换能器超声温度场的影响。研究结果表明曲面阵列相控聚焦换能器能有效地减少皮肤处的温升,对皮肤的伤害较小;对于曲面阵列相控聚焦换能器,不同占空比的正弦激励函数形成的可治疗区域(60℃以上)大小差别不大,但最高温度不同;随着频率升高,形成的可治疗区域体积减小;随着输入声强的增大,可治疗区域变大,但焦距不变。     

藏鸡微卫星文库的构建与微卫星标记筛选

通过磁珠富集法构建了藏鸡基因组微卫星富集文库,分离微卫星序列并对其进行分析.将藏鸡基因组DNA经Sau3AI酶切后纯化回收,连接特定接头.用生物素标记的(CA)12探针与藏鸡基因组酶切回收片段杂交,捕获200～900 bp片段,随后将获得的片段连接到pMD 18-T载体上,转化至JM109中,成功构建藏鸡微卫星富集文库.从1200个转化子中获得了353个阳性克隆,随机挑选53个测序,根据测序结果成功设计了18对藏鸡微卫星引物,最终筛选出6个具有多态性的微卫星标记,其PIC值均大于0.5,同时可以用于研究藏鸡遗传多样性.实验结果表明磁珠富集法能够有效提高分离微卫星标记的效率.     

复合诱变在青霉高产菊粉酶菌株选育中的应用

以Penicillium sp．B01为出发菌株,经吖黄素或DES（硫酸二乙酯）分别与^60Co-γ射线对其孢子悬液进行复合诱变。经过初筛和复筛,在30μg／mL吖黄素诱变时间2h,剂量率为4．11Gy／min的^60Co-γ射线辐射使累计剂量为20．55Gy复合诱变的条件下,筛选出一株菊粉酶活比出发菌株高32％的突变菌株B01-A13-Co31。经同工酶电泳验证,变异株与出发菌株相比酶带有所变化。将此菌种连续传代6次进行产酶性能的稳定性测定,表明此菌株具有良好的遗传稳定性。     

海洋细菌Bacillus sp．HN07的分离鉴定及所产碱性纤维素酶性质研究

从渤海海域（121°30′00″E,40°25′00″N）海泥中筛选到一株产碱性纤维素酶的海洋细菌。通过形态观察、生理生化特性及16SrDNA序列分析,鉴定该菌株属于芽孢杆菌属（Bacillus）,命名为Bacillussp．HN07。研究表明：HN07最适生长温度30℃,适宜在pH7．0～8．0、含2．0％～3．0％NaCl的培养条件下生长和产酶,并且具有较好的遗传稳定性。酶学性质初步研究显示,HN07所产碱性纤维素酶最适反应温度为45℃,最适pH为8．0,在碱性条件下具有较好的稳定性,具有潜在的工业应用价值。     

A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons

Recoding mechanisms are programmed protein synthesis events used commonly by viruses but only very rarely in cells for cellular gene expression. For example, HIV-1 has an absolute reliance on frameshifting to produce the correct ratio of key proteins critical for infectivity. To exploit such recoding sites as therapeutic targets, a simple homogeneous assay capable of detecting small perturbations in these low-frequency (<5%) events is required. Current assays based on dual luciferase reporters use expensive substrates and are labor-intensive, both impediments for high-throughput screening. We have developed a cell-based bifluorophore assay able to measure accurately small recoding changes (<0.1%) with a high Z′-factor in 24- or 96-well formats that could be extended to 384 wells. In cases of nonsense mutations arising within coding regions of genes, the assay is suitable for assessing the potential of screened compounds to increase read-through at these nonprogrammed stop signals of variable termination efficiency.     

Reduction of HDL levels lowers plasma PLTP and affects its distribution among lipoproteins in mice

Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1^−/−). Parallel to the strong reduction in PLTP activity in plasma of ABCA1^−/− mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1^−/− mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1^−/− mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1^−/−, apoE^−/− and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1^−/−, apoE^−/− and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4- , 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.     

A combination of ultrahigh throughput PathHunter and cytokine secretion assays to identify glucocorticoid receptor agonists

The use of ultrahigh throughput screens (uHTS) is a well-accepted mechanism to identify agonists and antagonists of target receptors. We used the Path Hunter [Path Hunter technology is a registered trademark of DiscoveRx Corporation.] technology from DiscoveRx to screen the entire Merck compound library for glucocorticoid receptor (GR) agonists in a 2.2-μl total reaction volume assayed in a 3456-well plate format. This single addition, homogenous assay which utilizes the principle of enzyme fragment complementation (EFC) to detect nuclear translocation of GR, an initial step of receptor activation, was used to successfully screen a large library of small molecules as indicated by an average signal to background ratio of approximately 4-fold and an average Z-factor value of 0.45. Hits from the HTS campaign were studied in a cytokine secretion assay in primary human monocytes to gain functional information regarding these compounds in a phenotypic and physiologically relevant setting. Our data indicate that using the PathHunter assay, we successfully identified compounds that showed agonism for the GR receptor in primary human monocytes and due to their performance in a physiologically relevant model they likely will have a better chance to evoke clinical efficacy.