Gene Expression Signature-Based Screening Identifies New Broadly Effective Influenza A Antivirals

Classical antiviral therapies target viral proteins and are consequently subject to resistance. To counteract this limitation, alternative strategies have been developed that target cellular factors. We hypothesized that such an approach could also be useful to identify broad-spectrum antivirals. The influenza A virus was used as a model for its viral diversity and because of the need to develop therapies against unpredictable viruses as recently underlined by the H1N1 pandemic. We proposed to identify a gene-expression signature associated with infection by different influenza A virus subtypes which would allow the identification of potential antiviral drugs with a broad anti-influenza spectrum of activity. We analyzed the cellular gene expression response to infection with five different human and avian influenza A virus strains and identified 300 genes as differentially expressed between infected and non-infected samples. The most 20 dysregulated genes were used to screen the connectivity map, a database of drug-associated gene expression profiles. Candidate antivirals were then identified by their inverse correlation to the query signature. We hypothesized that such molecules would induce an unfavorable cellular environment for influenza virus replication. Eight potential antivirals including ribavirin were identified and their effects were tested in vitro on five influenza A strains. Six of the molecules inhibited influenza viral growth. The new pandemic H1N1 virus, which was not used to define the gene expression signature of infection, was inhibited by five out of the eight identified molecules, demonstrating that this strategy could contribute to identifying new broad anti-influenza agents acting on cellular gene expression. The identified infection signature genes, the expression of which are modified upon infection, could encode cellular proteins involved in the viral life cycle. This is the first study showing that gene expression-based screening can be used to identify antivirals. Such an approach could accelerate drug discovery and be extended to other pathogens.     

Sample preparation project for the subcellular proteome of mouse liver

Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.     

Determination of the DNA methylation level of the marbled crayfish: an increase in sample throughput by an optimised sample preparation

Using a previously described capillary electrophoretic method with laser-induced fluorescence detection the genomic methylation level can be determined exactly. We present a sample preparation that eliminates the surplus of fluorescence marker used for coupling resulting in an increase of sample throughput from 75 to 250 analyses per week. The sensitivity of the method was also increased, which allows the determination of methylation levels under 1%. With these changes in sample preparation a methylation level of 1.64+/-0.03% in hepatopancreas DNA of the recently discovered marbled crayfish could be determined.     

The Successful Diagnosis and Typing of Systemic Amyloidosis Using A Microwave-Assisted Filter-Aided Fast Sample Preparation Method and LC/MS/MS Analysis

Laser microdissection followed by mass spectrometry has been successfully used for amyloid typing. However, sample contamination can interfere with proteomic analysis, and overnight digestion limits the analytical throughput. Moreover, current quantitative analysis methods are based on the spectrum count, which ignores differences in protein length and may lead to misdiagnoses. Here, we developed a microwave-assisted filter-aided sample preparation (maFASP) method that can efficiently remove contaminants with a 10-kDa cutoff ultrafiltration unit and can accelerate the digestion process with the assistance of a microwave. Additionally, two parameters (P- and D-scores) based on the exponentially modified protein abundance index were developed to define the existence of amyloid deposits and those causative proteins with the greatest abundance. Using our protocol, twenty cases of systemic amyloidosis that were well-typed according to clinical diagnostic standards (training group) and another twenty-four cases without subtype diagnoses (validation group) were analyzed. Using this approach, sample preparation could be completed within four hours. We successfully subtyped 100% of the cases in the training group, and the diagnostic success rate in the validation group was 91.7%. This maFASP-aided proteomic protocol represents an efficient approach for amyloid diagnosis and subtyping, particularly for serum-contaminated samples.     

Ecological Implications of an Improved Direct Viable Count Method for Aquatic Bacteria

The direct viable count method first described by Kogure et al. (Can. J. Microbiol. 25:415-420, 1979) was improved by using an antibiotic cocktail instead of nalidixic acid alone. We screened 100 marine isolates from two coastal areas for their sensitivities to five replication-inhibiting antibiotics, including four quinolones (nalidixic, piromidic, and pipemidic acids and ciprofloxacin) and one (beta)-lactam (cephalexin). It was shown that growth inhibition of all isolates cannot be readily achieved by using a single antibiotic. Inhibition was much more efficient when all the antibiotics were combined, making it possible to use this method with natural communities. In combination, the concentration of each antibiotic could be lowered and the incubation time could be increased without any growth. Under such conditions, it was shown that the fraction of substrate-responsive cells within natural marine communities is much greater (1 to 2 orders of magnitude) than those reported by traditional procedures. Furthermore, the new procedure made substrate-responsive cells more clearly distinguishable. These improvements resulted in an increased incubation time and were related to metabolic expression of slow-growing cells and/or to the recovery of starved cells. The increased fraction of viable cells within marine communities has ecological implications on the metabolic role of nonculturable cells.     

Trace elements in raw materials and nutrient media for cultivation of antibiotic-producing organisms

The contents of microelements such as Fe, Cu, Zn and As in samples of raw materials including food grade ones used in preparation of nutrient media for cultivation of antibiotic-producing organism and water were determined spectrophotometrically with using complex forming organic reagents. An original procedure for the sample combustion in the assay was developed. The natural background levels of the microelements in the nutrient media used in manufacture of some antibiotics were estimated.     

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2–3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.     

In vitro activity of honey,total alkaloids of Sophora alopecuroides and matrine alone and in combination with antibiotics against multidrug-resistant Pseudomonas aeruginosa isolates

Natural products, including honey, total alkaloids of Sophora alopecuroides (TASA) and matrine have been used in combination with antibiotics against various pathogenic bacteria. However, there are limited data on the antibacterial activity of these natural products in combination against multidrug-resistant Pseudomonas aeruginosa strains. The in vitro activity of honey, TASA and matrine alone and in combination with antibiotics against P. aeruginosa isolates was investigated. In this study, four biofilm-producing P. aeruginosa isolates, which were resistant to multiple antibiotics, were used. These natural products were not the most effective single agent against four isolates. The fractional inhibitory concentration index method revealed the synergistic effect of matrine and TASA-honey in combination with ciprofloxacin (Cip) against all tested isolates. When these combinations were used, the resistance of isolates to Cip was decreased significantly (six to eightfold reduction in the minimum inhibitory concentration of Cip. The disk diffusion method showed that all isolates were resistant to β-lactams. Combinations of these antibiotics with TASA and matrine changed slightly the activity of either antibiotic used as a single agent. All isolates produced metallo-β-lactamase enzymes (MBL). Pretreatment isolates with Cip-matrine and Cip-TASA-honey resulted in a statistically downregulated expression of the mexA gene. These natural products can be used against overactivating MexAB-OprM but not MBL-producing P. aeruginosa isolates.     

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2–3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.     

Determination of antibiotic EC50 using a zero‐flow microfluidic chip based growth phenotype assay

Current existing assay systems for evaluating antimicrobial activity suffer from several limitations including excess reagent consumption and inaccurate concentration gradient preparation. Recently, microfluidic systems have been developed to provide miniaturized platforms for antimicrobial susceptibility assays. However, some of current microfluidic based assays require continuous flows of reagents or elaborate preparation steps during concentration preparation. In this study, we introduce a novel microfluidic chip based growth phenotype assay that automatically generates a logarithmic concentration gradient and allows observing the growth of pathogenic bacteria under different concentrations of antibiotics in nanoliter batch culture reactors. We chose pathogen bacterium Pseudomonas aeruginosa as a model strain and evaluated the inhibitory effects of gentamicin and ciprofloxacin. We determined the EC₅₀ values and confirmed the validity of the present system by comparing the EC₅₀ values obtained through conventional test tube method. We demonstrated that the EC₅₀ values acquired from present assay are comparable to those obtained from conventional test tube cultures. The potential application of present assay system for investigating combinatorial effects of antibiotics on multidrug resistant pathogenic bacteria is discussed and it can be further used for systematic evaluation of antifungal or antiviral agents.     

围手术期不同处理因素对SD大鼠肠道菌群的影响

目的：探讨抗生素、肠道准备以及饮食对SD大鼠肠道茵群的影响。方法：将36只SD大鼠随机分为6组,分别采用不同的处理措施,分为对照组,抗生素组,肠道准备组,禁饮食组,术后早期禁饮食组和术后早期进食组,共处理4天,第5天脱颈椎处死,无茵条件下取回盲部粪便进行细菌培养并计数。结果：抗生素组、肠道准备组以及禁饮食组与对照组比较,抗生素组与肠道准备组、禁饮食组比较,大肠杆菌、类杆菌数量均明显增加,双歧杆菌、肠球菌数量均显著减少,双歧杆菌／大肠杆菌比值显著降低;其差异有统计学意义（P〈0．05）;术后早期进食组与术后禁饮食组相比,大肠杆茵、类杆菌数量有所减少,双歧杆菌、肠球菌数量有所增加,双歧杆菌／大肠杆菌比值有所提高,且其差异有统计学意义（P〈0．05）。结论：抗生素、肠道准备以及禁饮食均可引起SD大鼠肠道茵群失调,其中抗生素对肠道茵群的影响最大;并且术后早期进食对肠道茵群失调有改善作用。     

iTRAQ analysis of a cell culture model for malignant transformation, including comparison with 2D-PAGE and SILAC

To study human cancer development, cell culture models for malignant transformation can be used. In 1999 Hahn and Coworkers introduced such a model system and established herewith a basis for research on human tumorigenesis. Primary human fibroblasts are sequentially transduced with defined genetic elements (hTERT, SV40 ER, and H-RasV12), resulting in four defined cell lines, whereby the last has a fully transformed phenotype. In order to get a deeper insight into the molecular biology of human tumorigenesis, we compared the proteomes of these four cell lines following a multimethod concept. At the beginning we assumed SILAC and sample fractionation with COFRADIC is the method of choice to analyze the cell culture model for malignant transformation. Here, the compared samples are combined before sample preparation, thus avoiding differences in sample preparation, and using COFRADIC notably reduces sample complexity. Because 2D-PAGE is a standard method for the separation and visualization of closely related proteomes, we decided to analyze and compare the proteomes of these four cell lines in a first approach by differential 2D-PAGE. Surprisingly, we discovered much more unique results with iTRAQ and sample fractionation with SCX than with the combination of 2D-PAGE and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies not only in number of yielded results but also in analysis time. Here, we present the iTRAQ quantification results and compare them with the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the protein level at each transition. Thereby, SV40 has the strongest impact on the proteome. In detail we identified 201 regulated proteins. Beside others, these proteins are involved in cytoskeleton, RNA processing, and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins. For example, MCM proteins are up-regulated and collagens are down-regulated due to SV40 ER expression. Furthermore we made the observation that proteins containing the same domain have analogous regulation profiles during malignant transformation. For instance, several proteins containing a CH or LIM domain are down-regulated. Moreover, by this study and the defined cell culture model, changes could be clearly matched to specific steps during tumorigenesis.     

Antibiograms of pathogenic bacteria isolated from laboratory rabbits in Ibadan, Nigeria

Antibiotic sensitivity patterns of 21 bacterial isolates from some clinically ill New Zealand rabbits were evaluated against 12 commonly used antibiotics by the Kirby-Bauer disk-diffusion sensitivity testing method. The 21 isolates consisted of six Bordetella bronchiseptica, eight Pasteurella multocida, four Staphylococcus aureus and three Pseudomonas alcaligenes that were associated with snuffles, pneumonia, otitis media, genital infections and conjunctivitis in these groups of caged rabbits. The four bacteria species were all sensitive to kanamycin, gentamycin and enrofloxacin, with variable sensitivity to the other antibiotics tested. The results of this antibiogram could serve as a field guide in the treatment of very acute bacterial diseases of rabbit prior to the availability of the results of local sensitivity tests. Such sensitivity tests should be reviewed yearly to update this antibiogram.     

Usage of electrostatic eliminator reduces human keratin contamination significantly in gel-based proteomics analysis

In the field of bottom-up proteomics, heavy contamination of human keratins could hinder the comprehensive protein identification, especially for the detection of low abundance proteins. In this study, we examined the keratin contamination in the four major experimental procedures in gel-based proteomic analysis including gel preparation, gel electrophoresis, gel staining, and in-gel digestion. We found that in-gel digestion procedure might be of importance corresponding to keratin contaminants compared to the other three ones. The human keratin contamination was reduced significantly by using an electrostatic eliminator during in-gel digestion, suggesting that static electricity built up on insulated experimental materials might be one of the essential causes of keratin contamination. We herein proposed a series of methods for improving experimental conditions and sample treatment in order to minimize the keratin contamination in an economical and practical way.     

A note on inhibition test and electrophoretic detection limits of antibiotics used in British animal husbandry

A four plate microbiological inhibition test (the FPT) and a bioelectrophoretic method were evaluated for their ability to detect a range of antibiotic agents, which may be present as residues in animal tissues following their therapeutic use in animal husbandry. Both methods exhibited a wide range of sensitivities and several of the tested antibiotics could not be detected by either method. The pattern of responses across the bacterial plates in the FPT could not be used to identify agents and the bioelectrophoretic inhibition zone diameters were generally too large to allow the use of R_s values for identification. The Bacillus subtilis pH 7.2 plate with trimethoprim added was as effective as the four bacterial plates used in the FPT in antibiotic detection.     

A comparison of alternative sample preparation procedures for the analysis of swainsonine using LC‐MS/MS

Introduction – Swainsonine, a polyhydroxy indolizidine alkaloid and known glycosidase inhibitor, is found in a number of different plants that cause a lysosomal storage disease known as locoism in the western USA. Most recently swainsonine has been analysed by LC‐MS/MS after sample extraction and preparation from ion‐exchange resins. Objective – To compare previously published sample preparation procedures with several new alternative procedures to provide methods using either commercially available solid‐phase extraction equipment or procedures which significantly reduce sample preparation time. Methodology – A previously reported and validated sample preparation method using ion‐exchange resin was compared with methods using a commercially available solid‐phase extraction cartridge, a solvent partitioning procedure or a single solvent extraction procedure using one of two solvents. Twenty different plant samples of varying swainsonine concentrations were prepared in triplicate and analysed by LC‐MS/MS. The measured concentration of swainsonine was then statistically compared between methods. Results – There were no statistically significant differences found between four of the five different sample preparation methods tested. Conclusion – A commercially available SPE cartridge can be used to replace the previously used ion‐exchange resin for swainsonine analysis. For very rapid analyses the SPE procedure can be eliminated and a simple, single solvent extraction step used for sample preparation. Published in 2010 by John Wiley & Sons, Ltd.     

An improved method for the observation and enumeration of heterotrophic and photoautotrophic microplankton

Fluorescence microscopy is a preferred technique for discriminating between heterotrophic and photoautotrophic microorganisms in plankton studies. An improved technique is described which employs sample concentration by filtration onto Nuclepore filters followed by transfer to a glycerine jelly preparation for identification of phytoplankton by autofluorescence of photosynthetic pigments and examination of all microorganisms by phase-contrast microscopy. The method is rapid, and has been used routinely at sea to provide semipermanent preparations of pico-and nanoplankton including naked flagellates for subsequent enumeration onshore.     

Observations below multiple lower limits of quantification: How to estimate the mean and variance

Multiple lower limits of quantification (MLOQs) result if various laboratories are involved in the analysis of concentration data and some observations are too low to be quantified. For normally distributed data under MLOQs there exists only the multiple regression method of Helsel to estimate the mean and variance. We propose a simple imputation method and two new maximum likelihood estimation methods: the multiple truncated sample method and the multiple censored sample method. A simulation study is conducted to compare the performances of the newly introduced methods to Helsel's via the criteria root mean squared error (RMSE) and bias of the parameter estimates. Two and four lower limits of quantification (LLOQs), various amounts of unquantifiable observations and two sample sizes are studied. Furthermore, the robustness is investigated under model misspecification. The methods perform with decreasing accuracy for increasing rates of unquantified observations. Increasing sample sizes lead to smaller bias. There is almost no change in the performance between two and four LLOQs. The magnitude of the variance impairs the performance of all methods. For a smaller variance, the multiple censored sample method leads to superior estimates regarding the RMSE and bias, whereas Helsel's method is superior regarding the bias for a larger variance. Under model misspecification, Helsel's method was inferior to the other methods. Estimating the mean, the multiple censored sample method performed better, whereas the multiple truncated sample method performs best in estimating the variance. Summarizing, for a large sample size and normally distributed data we recommend to use Helsel's method. Otherwise, the multiple censored sample method should be used to obtain estimates of the mean and variance of data including MLOQs.     

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed.     

Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.