A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target

As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.     

A novel strategy for construction of immuno-PCR gene probe and its preliminary application in diagnosis

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A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector

siRNA technique has been widely used to study the gene functions and to develop disease therapeutics. One of the challenges of RNAi application is to obtain the most effective target specific siRNA sequences. Currently the process to select and validate optimal siRNA sites for a given gene, which is usually based on screening by using Western blot, Northern blot or Q-PCR, remains empirical and time consuming. Although few fluorescence-based siRNA sequence selection systems have proven useful, the rapid and efficient screening of siRNA target sites is still challenging. In the paper, we developed a quick and efficient method to screen siRNA target sites with a novel single vector system, which contains the following cassettes: (1) an eGFP reporter gene expression cassette followed by a multiple cloning site and SV40 pA for insertion of a target sequence; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions; and (3) an internal loading control, mCherry reporter gene. Based on this one-step transfection with single vector system, we could rapidly screen effective, target specific siRNA fragments in an unbiased manner by judging the fluorescence intensity ratio of eGFP to mCherry. The generation of this novel vector system will promote the application of siRNA in basic research and disease therapy.     

小黄花茶内生真菌的多样性分析及抑菌活性初筛

为探究小黄花茶内生真菌种类和种群分布规律以及对植物病原真菌的抑制作用,该研究采用组织分离法对小黄花茶内生真菌进行分离纯化,基于形态学和分子生物学进行鉴定并结合统计学分析评价其多样性,再通过平板对峙法筛选出具有抑菌活性的菌株。结果表明：(1)从小黄花茶324份组织块中分离得到内生真菌261株,隶属1门5纲9目22属,其中优势属包括炭疽菌属(Colletotrichum)、间座壳属(Diaporthe)、拟盘多毛孢属(Pestalotiopsis),分离频率分别为21.84%、16.86%、10.34%。(2)研究发现小黄花茶内生真菌在不同季节分布不同,冬季分离出的菌株数量最多,为72株(占27.59%,隶属16个属),春季62株(隶属13个属),夏季59株(隶属15个属),秋季68株(隶属13个属),冬季的香农-维纳指数(H′)、辛普森指数(D)、Pielou’s均匀度指数(E)和Margalef’s丰富度指数(M)最高,春季与冬季内生真菌种类相似性较高,夏季与秋季内生真菌种类相似性较高。(3)小黄花茶内生真菌不同部位分布不同,茎中内生真菌的分布最多,有102株(占39.08%,隶属15...     

A novel genomic approach identifies bacterial DNA-dependent RNA polymerase as the target of an antibacterial oligodeoxynucleotide, RBL1

Rapid emergence of antibiotic-resistant bacterial pathogens limits the applicability of existing drugs, which has created an urgent need for novel antibiotics preferably with entirely new mechanisms of action. Oligodeoxynucleotides (ODNs) and their modified forms have been shown to inhibit bacterial gene expression, representing a potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline (Tet)-inducible, randomized single-stranded DNA (ssDNA) expression library was constructed and screened for conditional growth-defective or lethal phenotypes in an Escherichia coli system. From approximately 5000 transformants screened, 12 bacterial colonies were identified with either growth-defective or lethal phenotypes. One clone, CY01, with a lethal phenotype was selected and sequenced, and the ODN sequence that it generates was designated as RBL-1. Because RBL-1 shows no significant homologies to any bacterial gene sequence, a potential RBL-1 targeting protein was isolated by affinity purification. Using mass spectrometry analysis, this protein was identified as bacterial DNA-dependent RNA polymerase (RNAP). RBL-1 was further shown to effectively inhibit RNA polymerase activity in vitro. The usage of this randomized ssDNA expression library screening technology to selectively modulate production and/or function of proteins may provide a powerful strategy in both identifying novel gene targets for antibiotic discovery and developing novel antibacterial agents.     

A rapid novel strategy for screening of antibody phage libraries for production,purification, and functional characterization of amber stop codons containing single-chain antibody fragments

Phage display antibody (PDA) libraries, allows the rapid isolation and characterization of high specificity monoclonal antibodies for therapeutic and diagnostic applications. However, selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias towards clones containing randomly generated amber stop codons, complicating the identification of high affinity binding antibodies. We screened Tomlinson I and J library against receptor binding domain (RBD) of SARS CoV2, eight clones which showed positive binding in phage ELISA, contained one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences. The presence of amber stop codons within the antibody sequence causes the premature termination of soluble form of scFv expression in nonsuppressor Escherichia coli strain. In the present study, we have used a novel strategy that allows soluble expression of scFvs having amber stop codon in their gene sequences (without phage PIII protein fusion), in the suppressor strain. This strategy of introduction of Ochre (TAA) codon at the junction of scFv and PIII gene, speeds up the initial screening process which is critical for selecting the right scFvs for further studies. Present strategy leads to the identification of a scFv, B8 that binds specifically with nanomolar affinity toward SARS CoV 2 RBD, which otherwise lost in terms of traditional methodology.     

A simple strategy for screening for glucose intolerance, using glycated haemoglobin, in individuals admitted with acute coronary syndrome

Glucose intolerance is common but often remains undiagnosed and untreated in people with acute coronary syndrome. The best approach to screening for glucose intolerance post-acute coronary syndrome remains debated. The World Health Organization has recently advocated the use of HbA(1c) in diagnosing Type 2 diabetes. A screening strategy using HbA(1c) as the preferred test would be pragmatic and improve early detection and management of glucose intolerance in acute coronary care practice. In this commentary, we discuss the relevant literature and guidelines in this area and propose a simple and pragmatic algorithm based on the use of HbA(1c) to screen for glucose intolerance during and after admission with acute coronary syndrome.     

A novel approach for affinity-based screening of target specific ligands: application of photoreactive D-glyceraldehyde-3-phosphate dehydrogenase

A novel application of the photoaffinity technique has been developed for the efficient discovery of small ligand and macromolecule interaction. The approach, photoaffinity capture, uses a photoreactive protein together with immobilized ligand for the rapid screening of competitive inhibitors. The set of photoreactive glyceraldehyde-3-phosphate dehydrogenase (photo-GAPDH) and immobilized dye ligand was prepared and examined as a model system. The photo-GAPDH was shown to efficiently capture the immobilized ligand. When nonimmobilized competitive ligands were included in the system, the capture was prevented in accordance with the affinity of the ligands. The present approach would provide an efficient tool for affinity-based screening of ligand libraries.     

A novel microarray strategy for detecting genes and pathways in microbes with unsequenced genomes

Expression profile analysis of genes provides valuable information concerning the genetic response of cells to stimuli. We describe an adaptation of this technology that can be used to probe for the expression of specific families of genes in microbial species. In our method a combination of sets of oligonucleotide probes representing fingerprint sequences specific to protein families is used to identify the presence and expression levels of family homologs in a microbial cell. We demonstrate computationally, using exemplars, that when the cDNA complement from an organism is sequentially screened against a set of specific motif oligonucleotides, statistically significant information can be obtained concerning the expression of the corresponding genes. This method can be used to identify specific genes and pathways simultaneously in several organisms of interest even in the absence of sequence information from the organisms.     

A new strategy for efficient in vivo screening of mutagenized Drosophila embryos

The analysis of mutants is an indispensable approach towards characterizing gene function. Combining several tools of Drosophila genetics, we designed a new strategy for a mutagenesis screen which is fast, easy-to-apply, and cheap. The combination of a cell-specific Gal4 line with an upstream activating sequence-green fluorescent protein (UAS-GFP) allows the in vivo detection of the cells or tissues of interest without the need for fixation and staining. To further simplify and accelerate the screening procedure, we generated recombinant flies that carry the Gal80 transgene in balancer chromosomes. Gal80 inactivates Gal4; and thus prevents GFP-expression during embryonic and postembryonic development in all individuals carrying the balancer chromosomes. This allows for an easy distinction in vivo between heterozygous and homozygous mutants, the latter being the only ones expressing GFP. Since most of the fly strains and balancer chromosomes can be substituted, this method is suitable for nearly any mutagenesis screen that does not have major restrictions.     

氮磷营养盐影响海水浮游硅藻种群组成的初步研究

用实验生态学方法研究了氮磷营养盐对近岸海洋硅藻组成的影响?结果表明,氮磷营养盐浓度及其比例可能对海水 游硅藻组成有着明显的影响,氮磷营养盐浓度越高,氮磷化离Ｒｅｄｆｉｅｌｄ比越远,硅藻种类越少,Ｓｈａｎｎｏｎ指数越低,这一结果在对虾养殖池浮游植物的观测中得到初步验证。     

A strategy for rapid production and screening of yeast artificial chromosome libraries

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture pools, and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4–5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the 1(I) collagen (Colla1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.     

A novel and simple method of screening compounds for interaction with DNA: A validation study

We report the development of a simple, cost-effective assay for detecting compounds that have the ability to interact with and modify DNA. Potential uses for the assay lie in the areas of early genotoxicity testing of drug candidates, anticancer and antibiotic drug discovery, environmental monitoring and testing in the food, beverage and cosmetics industries. At present the assay has been used to assess direct-acting compounds only and it is yet to be established whether the assay is compatible with bio-activation. The methodology is based on the oxidative reaction of potassium permanganate with pyrimidine bases, which have become perturbed and more reactive by the agent under test. Results are recorded by use of UV/vis spectroscopy. The adaptation to a multi-well plate format provides the capacity for high throughput utilizing small amounts of compounds. Over 100 compounds, comprising different classes of DNA-binding chemicals as well as non-binding controls, have been put through the assay and the results compared with existing genotoxicity testing data from other methods. The assay has shown to be predictive of the results of other genotoxicity testing methods. We have found that the method is overall predictive of 71% of Ames bacterial reverse-mutation test results (where data are given) encompassing both negative and positive results.     

A screening strategy for heterologous protein expression in Escherichia coli with the highest return of investment

Heterologous protein expression in Escherichia coli is commonly used to obtain recombinant proteins for a variety of downstream applications. However, many proteins are not, or are only poorly, expressed in soluble form. High level expression often leads to the formation of inclusion bodies and an inactive product that needs to be refolded. By screening the solubility pattern for a set of 71 target proteins in different host-strains and varying parameters such as location of purification tag, promoter and induction temperature we propose a protocol with a success rate of 77% of clones returning a soluble protein. This protocol is particularly suitable for high-throughput screening with the goal to obtain soluble protein product for e.g. structure determination.     

A gold nanoparticle-based strategy for label-free and colorimetric screening of DNA triplex binders

A label-free colorimetric assay, using non-crosslinking AuNP aggregation, has been developed for the screening of specific triplex DNA binders. The relative binding affinities can be simultaneously determined. Our novel assay is simple in design and fast in operation, avoiding either AuNPs modification or oligonucleotide labeling, and easy to implement for visual detection. This strategy may offer a new approach for developing low cost, sensitive and high-throughput screening platform that is likely to be highly useful in a wide range of applications.