An efficient molecular docking method for adsorbent screening

In this study, with flavonol glycosides (FG) and terpene lactones (TL) in ginkgo biloba extract (GBE) as the targets for separation, we investigated the effectiveness of molecular docking in adsorbent screening. Several polyamine-modified methyl acylate-co-divinylbenzene (MA-co-DVB) adsorbent models were built, and their affinity to rutin, quercetin and ginkgolide B (GB) was evaluated via molecular docking. The model of ethylenediamine-modified adsorbent showed the largest difference in affinity between to GB and to quercetin as well as rutin, and thus this adsorbent could have the best separation performance. The results of the subsequently conducted static adsorption and dynamic adsorption experiments correlated well with docking results. Finally, using ethylenediamine-modified MA-co-DVB adsorbent, nearly complete separation of the FG and TL in GBE was simply achieved by one step of adsorption-desorption. Thus, the reported molecular docking method is expected to be helpful for rapid adsorbent screening.     

An efficient method for in vitro fertilization in rabbits

Abstract

This experiment was carried out to study a simple and efficient method for in vitro production of rabbit embryos. Newly ejaculated rabbit spermatozoa were used to fertilize superovulated oocytes after capacitation in vitro with four different media: (A) isotonic defined medium (DM)+heparin, (B) DM only,(C) DM+ high ionic strength defined medium (HIS), and (D) DM supplemented with 10mM NaHCO₃ (mDM) +HIS supplemented with 10mM NaHCO₃ (mHIS). The presumptive zygotes were cultured in M199 supplemented with 10% FCS, 1.25mM Na Pyruvate and 0.1mM EDTA (mM199). The cleavage rates after 24h of incubation were 29.3%, 32.1%, 64.9%, and 91.6% respectively, and the rates of blastocyst formation after 72h were 0, 27.3%, 58.4% and 85.2%, respectively. The results in the (D) treatment were significantly better than the other three treatments (p<0.01). Developmental potential of in vivo and in vitro derived zygotes was also compared using the mM199. The percentages of blastocyst and hatching blastocyst in the two groups were 92.5% and 87.2% after 84h, and 84.9% and 83.7% after 108h, respectively, and the two groups were not significantly different (p>0.05). The developmental progress of the two groups was nearly synchronous towards the end of culture. When IVF embryos from 2‐ to 4‐cell stage were transferred into recipients, the pregnancy rate did not differ from in vivo fertilization, but the rate of live young from IVF was significantly lower than from in vivo. The results of this experiment showed that ejaculated rabbit sperm could be capacitated efficiently after treatment of mDM and mHIS, and rabbit IVF embryos achieved great development in mM199 in vitro.     

An efficient method for in vitro fertilization in rabbits

This experiment was carried out to study a simple and efficient method for in vitro production of rabbit embryos. Newly ejaculated rabbit spermatozoa were used to fertilize superovulated oocytes after capacitation in vitro with four different media: (A) isotonic defined medium (DM)+heparin, (B) DM only,(C) DM+ high ionic strength defined medium (HIS), and (D) DM supplemented with 10mM NaHCO3 (mDM) +HIS supplemented with 10mM NaHCO3 (mHIS). The presumptive zygotes were cultured in M199 supplemented with 10% FCS, 1.25mM Na Pyruvate and 0.1mM EDTA (mM199). The cleavage rates after 24h of incubation were 29.3%, 32.1%, 64.9%, and 91.6% respectively, and the rates of blastocyst formation after 72h were 0, 27.3%, 58.4% and 85.2%, respectively. The results in the (D) treatment were significantly better than the other three treatments (p<0.01). Developmental potential of in vivo and in vitro derived zygotes was also compared using the mM199. The percentages of blastocyst and hatching blastocyst in the two groups were 92.5% and 87.2% after 84h, and 84.9% and 83.7% after 108h, respectively, and the two groups were not significantly different (p>0.05). The developmental progress of the two groups was nearly synchronous towards the end of culture. When IVF embryos from 2- to 4-cell stage were transferred into recipients, the pregnancy rate did not differ from in vivo fertilization, but the rate of live young from IVF was significantly lower than from in vivo. The results of this experiment showed that ejaculated rabbit sperm could be capacitated efficiently after treatment of mDM and mHIS, and rabbit IVF embryos achieved great development in mM199 in vitro.     

堆肥中微生物总DNA的高效提取

采用化学裂解和酶解相结合的方法,选择加入PVPP的高盐缓冲液作为细胞裂解的反应体系,并以PEG-8000进行DNA沉淀,从高有机含量的堆肥样品中进行微生物总DNA的提取。结果表明,从4种性质不同的堆肥中均获得了高质量的微生物总DNA,所得的DNA分子片段在23kb左右;每克干重堆肥的总DNA提取量为63.54±12.08μg~106.50±28.36μg,A260/A280大于1.6,A260/A230大于1.8,不用经过纯化可以直接进行PCR扩增和限制性酶切;以该DNA为模板进行微生物区系的DGGE分析,显示了丰富的微生物多样性。该方法减少了通常环境样品DNA提取过程中的纯化步骤,减少了DNA的损失,为从事微生物分子生态学,尤其是那些针对高有机含量以及获取极为不易的环境样品的研究而言是十分有益的。     

An efficient platform for genetic selection and screening of gene switches in Escherichia coli

Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.     

小麦线粒体DNA的高效提取方法

以小麦黄化苗为材料, 通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体, 用SDS和蛋白酶K裂解线粒体, 经酚/氯仿抽提除去蛋白, 并用RNase A消化而得到单纯线粒体DNA(mtDNA)。对所提取的mtDNA进行紫外吸收光度分析, A₂₆₀/A₂₈₀ 平均为1.92, A260/A230 平均为2.09, 平均每克黄化苗可提取mtDNA 26.85 mg; 并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增, 均得到清晰的电泳图谱。结果表明: 此提取方法得到的mtDNA, 不但产率高、结构完整, 而且能有效去除核DNA、RNA和蛋白质等杂质, 获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现, 通过调整线粒体裂解温度(先50℃裂解1 h, 再37℃裂解1 h), 亦可大幅度提高mtDNA的产率。     

An efficient and non-destructive DNA extraction method for oribatid mites

Molecular methods play an important role in systematic acarology and DNA extraction is the first step in this ground. Nowadays, the varieties of methods are being used for extraction of DNA, but most of them destroy the important external characters that are essential for morphological identification. In order to overcome the problem of associating molecular and morphological information, we have developed a simple, efficient and non-destructive DNA extraction method for oribatid mites. The non-destructive method was tested on three different species [Oppia nitens C.L. Koch, 1836 (Oppiidae), Acrotritia ardua C.L. Koch, 1841 (Euphthiracaridae), and Amerus polonicus Kulczynski, 1902 (Ameridae)] and exoskeleton of specimens were preserved in Hoyer’s medium on glass microscopic slides for further identification via morphological studies. This method is more time consuming than commercial kits, but is easier and cheaper, meanwhile, allows systematic analysis with linking the morphological and genetic traits from a single mite. The most important advantage of TNES method is that after using this non-destructive method, specimens preserve all of their morphological features.     

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of totalgenomic DNA from 15 red algal species. It resulted in 0.1 g high quality DNAfrom 1 mg fresh algal material, with an A₂₆₀/A₂₈₀ratio of 1.68–1.90.Using this rapidly isolated DNA, the 18S ribosomal RNA genes (rDNA) and the nuclearribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. Thetested DNA was suitable for restriction endonuclease digestion, genetic markeranalysis and polymerase chain reaction (PCR) amplification, and may be valid forother genetic manipulation.     

An efficient haplotyping method with DNA pools

Determination of haplotype frequencies (the joint distribution of genetic markers) in large population samples is a powerful tool for association studies. This is due to their greater extent of polymorphism since any two bi-allelic single nucleotide polymorphisms (SNPs) generate a potential four-allele genetic marker. Therefore, a haplotype may capture a given functional polymorphism with higher statistical power than its SNP components. The statistical estimation of haplotype frequencies, usually employed in linkage disequilibrium studies, requires individual genotyping for each SNP in the haplotype, thus making it an expensive process. In this study, we describe a new method for direct measurement of haplotype frequencies in DNA pools by allele-specific, long-range haplotype amplification. The proposed method allows the efficient determination of haplotypes composed of two SNPs in close vicinity (up to 20 kb).     

一种快速高效提取病原真菌DNA作为PCR模板的方法

真菌rDNA-ITS序列分析适合于较高等级水平的生物群体间的系统分析。真菌DNA的提取采用传统的方法,步骤繁琐,需要较长时间。采用Chelex-100法提取真菌DNA,使用PCR扩增rDNA-ITS序列评价提取核酸的质量。结果显示,该方法具有经济、简便、快速、高效的特点,是一种比较理想的提取真菌基因组DNA作为PCR模板的方法。     

An efficient method for the detection and elimination of systematic error in high-throughput screening

MOTIVATION: High-throughput screening (HTS) is an early-stage process in drug discovery which allows thousands of chemical compounds to be tested in a single study. We report a method for correcting HTS data prior to the hit selection process (i.e. selection of active compounds). The proposed correction minimizes the impact of systematic errors which may affect the hit selection in HTS. The introduced method, called a well correction, proceeds by correcting the distribution of measurements within wells of a given HTS assay. We use simulated and experimental data to illustrate the advantages of the new method compared to other widely-used methods of data correction and hit selection in HTS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

An in vitro selection scheme for oligonucleotide probes to discriminate between closely related DNA sequences

Using an in vitro selection, we have obtained oligonucleotide probes with high discriminatory power against multiple, similar nucleic acid sequences, which is often required in diagnostic applications for simultaneous testing of such sequences. We have tested this approach, referred to as iterative hybridizations, by selecting probes against six 22-nt-long sequence variants representing human papillomavirus, (HPV). We have obtained probes that efficiently discriminate between HPV types that differ by 3–7nt. The probes were found effective to recognize HPV sequences of the type 6, 11, 16, 18 and a pair of type 31 and 33, either when immobilized on a solid support or in a reverse configuration, as well to discriminate HPV types from the clinical samples. This methodology can be extended to generate diagnostic kits that rely on nucleic acid hybridization between closely related sequences. In this approach, instead of adjusting hybridization conditions to the intended set of probe–target pairs, we ‘adjust’, through in vitro selection, the probes to the conditions we have chosen. Importantly, these conditions have to be ‘relaxed’, allowing the formation of a variety of not fully complementary complexes from which those that efficiently recognize and discriminate intended from non-intended targets can be readily selected.     

An in vitro nucleoside analog screening method for cancer gene therapy

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene therapy research. We have developed an in vitro screening assay to assess the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). The thymidine kinase gene enhances nucleoside phosphorylation to nucleotides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transfected 3T3-NIH). Two types of analysis are performed: a cytotoxicity assay, the neutral red uptake assay to assess the IC₅₀ on the two cell lines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic profiles after incubation of cells with tritiated analogs. Results show that cells expressing the HSV-TK gene are more sensitive than the parent cells to the effect of acyclovir or ganciclovir, the reference purine analog drugs, and also to the effect of pyrimidine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxyuridine. Promising nucleoside analogs for gene therapy that can be achieved by HSV-TK could be evaluated using this model.Abbreviations ACV acyclovir - ACV-MP acyclovir monophosphate - ACV-DP acyclovir diphosphate - ACV-TP acyclovir triphosphate - BDU bromodeoxyuridine - BVDU bromovinyldeoxyuridine - EDU ethyldeoxyuridine - FDU fluorodeoxyuridine - GCV ganciclovir - HSV-TK herpes simplex virus thymidine kinase gene - IDU iododeoxyuridine - NA nucleoside analog     

Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro

Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ?29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A(3) (GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.     

An efficient in vitro method for mass propagation of Potentilia potanii wolf

Summary This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and &#x03B1;-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mgl^&#x2212;1 BA and 1.0 mgl^&#x2212;1 NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mgl^&#x2212;1 NAA and 0.5 mgl^&#x2212;1 indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.     

An efficient method for the in vitro management of multiple garlic accessions

Summary A simple protocol for garlic in vitro management is described. It comprises a single medium for all developmental stages and genotypes tested, and the use of immature bulbs as source of axillary buds. Although genetic variability exists among the different accessions tested, for both the multiplication rate and bulblet size, acceptable values of multiplication were reached for all the accessions, and values of bulb formation approached 100% in the shoots produced.     

An efficient method for extracting plasma ions in laser isotope separation systems

The possibility of using a Hall-current accelerator to extract ions from a partially ionized plasma produced by selective laser isotope photoionization of atomic vapor is examined. A mechanism for ion acceleration is investigated using one-dimensional time-dependent equations of two-fluid magnetohydrodynamics. The current cutoff due to the ion space charge is prevented by electron emission. It is shown that, at an accelerating voltage of 25–50 V and emission current density of several mA/cm², the ion component is accelerated throughout the entire plasma volume up to a velocity of ～10⁵ cm/s in a few microseconds. The influence of resonant charge exchange and secondary ionization by electrons on both the acceleration dynamics and selectivity degradation is taken into account. It is shown that the Hall-current extractor allows one to avoid selectivity degradation even when the plasma size exceeds the charge-exchange mean free path by one order of magnitude.     

Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates

Sequence-specific pausing occurs during DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase holoenzyme in the presence of the T4 helix destabilizing protein (gene 32 protein). Two of the six strongest pause sites on a double-stranded bacteriophage fd DNA template are in regions where hairpin helices are predicted to form when the DNA is single stranded. However, the other pause sites are in regions that are not obviously involved in secondary structure. The positions of the DNA chain ends produced at one pause site of each type were determined to within +/- 2 nucleotides. At this resolution, a clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions. The addition of the T4 gene 41 protein (a DNA helicase that forms part of the T4 primosome) to the above replication system greatly increases the rate of fork movement and eliminates detectable pausing. In contrast, the addition of the T4 dda protein (a second DNA helicase that increases the rate of fork movement to a similar extent) has no affect on replication fork pausing. This difference could either be due to specific protein-protein interactions formed between the polymerase holoenzyme and the 41 protein or to the highly processive movement of the 41 protein along the displaced DNA strand.