Reliability analysis of the Ahringer <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> RNAi feeding library: a guide for genome-wide screens

Background

The Ahringer C. elegans RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function. However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1) mis-annotation (the clone with the retired gene name should be remapped to the actual target gene); 2) nonspecific PCR amplification; 3) cross-RNAi; 4) mis-operation such as sample loading error, etc.

Results

Here we performed a reliability analysis on the Ahringer C. elegans RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3%) of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54%) bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs). The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (http://biocompute.bmi.ac.cn/CelRNAi/) was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies.

Conclusions

Because of the potential unreliability of the Ahringer C. elegans RNAi feeding library, we strongly suggest the user examine the reliability information of the bacterial strains in the CelRNAi database before performing RNAi experiments, as well as the post-RNAi experiment analysis.

     

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10⁻⁵–11×10⁻⁵ yr⁻¹. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.     

Analysis and modeling of the variable region of camelid single-domain antibodies

Camelids have a special type of Ab, known as heavy chain Abs, which are devoid of classical Ab light chains. Relative to classical Abs, camelid heavy chain Abs (cAbs) have comparable immunogenicity, Ag recognition diversity and binding affinities, higher stability and solubility, and better manufacturability, making them promising candidates for alternate therapeutic scaffolds. Rational engineering of cAbs to improve therapeutic function requires knowledge of the differences of sequence and structural features between cAbs and classical Abs. In this study, amino acid sequences of 27 cAb variable regions (V(H)H) were aligned with the respective regions of 54 classical Abs to detect amino acid differences, enabling automatic identification of cAb V(H)H CDRs. CDR analysis revealed that the H1 often (and sometimes the H2) adopts diverse conformations not classifiable by established canonical rules. Also, although the cAb H3 is much longer than classical H3 loops, it often contains common structural motifs and sometimes a disulfide bond to the H1. Leveraging these observations, we created a Monte Carlo-based cAb V(H)H structural modeling tool, where the CDR H1 and H2 loops exhibited a median root-mean-square deviation to natives of 3.1 and 1.5 ?, respectively. The protocol generated 8-12, 14-16, and 16-24 residue H3 loops with a median root-mean-square deviation to natives of 5.7, 4.5, and 6.8 ?, respectively. The large deviation of the predicted loops underscores the challenge in modeling such long loops. cAb V(H)H homology models can provide structural insights into interaction mechanisms to enable development of novel Abs for therapeutic and biotechnological use.     

应用原子力显微镜分析猪脂肪前体细胞的分化

脂肪形成过程中发生的异常变化与许多疾病的产生有着密切的关系。为深入了解脂肪形成的机制,利用原子力显微镜研究脂肪前体细胞向成熟脂肪细胞分化前后细胞形貌、超微结构和机械性能的变化。结果表明,脂肪前体细胞与成熟脂肪细胞在形貌上存在明显的差异。在超微结构的探测中成熟脂肪细胞表面粗糙度低于脂肪前体细胞。通过力曲线的分析得出,分化前后两种细胞的机械性质均发生改变。脂肪前体细胞在粘弹力、硬度和杨氏模量的研究中比成熟脂肪细胞都高出约20％。原子力显微镜在纳米尺度上分析脂肪前体细胞向成熟脂肪细胞分化过程中细胞膜的改变,其研究结果为进一步探讨脂肪形成机制提供可视化定量数据。     

Fragment‐based modeling of membrane protein loops: Successes,failures, and prospects for the future

Membrane proteins (MPs) have become a major focus in structure prediction, due to their medical importance. There is, however, a lack of fast and reliable methods that specialize in the modeling of MP loops. Often methods designed for soluble proteins (SPs) are applied directly to MPs. In this article, we investigate the validity of such an approach in the realm of fragment‐based methods. We also examined the differences in membrane and soluble protein loops that might affect accuracy. We test our ability to predict soluble and MP loops with the previously published method FREAD. We show that it is possible to predict accurately the structure of MP loops using a database of MP fragments (0.5–1 Å median root‐mean‐square deviation). The presence of homologous proteins in the database helps prediction accuracy. However, even when homologues are removed better results are still achieved using fragments of MPs (0.8–1.6 Å) rather than SPs (1–4 Å) to model MP loops. We find that many fragments of SPs have shapes similar to their MP counterparts but have very different sequences; however, they do not appear to differ in their substitution patterns. Our findings may allow further improvements to fragment‐based loop modeling algorithms for MPs. The current version of our proof‐of‐concept loop modeling protocol produces high‐accuracy loop models for MPs and is available as a web server at http://medeller.info/fread . Proteins 2014; 82:175–186. © 2013 Wiley Periodicals, Inc.     

Single-cell paired-end genome sequencing reveals structural variation per cell cycle

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis.     

Characterization of molecular recognition of Phosphoinositide-3-kinase α inhibitor through molecular dynamics simulation

Phosphatidylinositol 3-kinase α (PI3Kα) is a promising target for anticancer drug discovery due to its overactivation in tumor cells. To systematically investigate the interactions between PI3Kα and PIK75 which is the most selective PI3Kα inhibitor reported to date, molecular docking, molecular dynamics simulation, and ensuing energetic analysis were utilized. The binding free energy between PI3Kα and PIK75 is −10.04 kcal•mol⁻¹ using MMPBSA method, while −13.88 kcal•mol⁻¹ using MMGBSA method, which is beneficial for the binding. The van der Waals/hydrophobic and electrostatic interactions play critical roles for the binding. The binding mode of PIK75 for PI3Kα is predicted. The conserved hydrophobic adenine region of PI3Kα made up of Ile800, Ile848, Val850, Val851, Met922, Phe930, and Ile932 accommodates the flat 6-bromine imidazo[1,2-a]pyridine ring of PIK75. The 2-methyl-5-nitrophenyl group of PIK75 extends to the P-loop region, and has four hydrogen-bond arms with the backbone and side chain of Ser773 and Ser774. And the distinct conformation of the P-loop induced by PIK75 is speculated to be responsible for the selectivity profile of PIK75. The predicted binding mode of PIK75 for PI3Kα presented in this study may help design high affinity and selective compounds to target PI3Kα.     

Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential

Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications.