Evaluation of a colorimetric method for vitamin A estimation

A simple colorimetric procedure for plasma vitamin A is evaluated, which does not require sophisticated or expensive equipment. Vitamin A values obtained with 30 human plasma samples and 11 liver samples obtained from lactating rabbits, using a colorimetric procedure based on Carr-Price reaction with ferric chloride and acetyl chloride were compared with those obtained with spectrophotometric and spectrofluorimetric micro methods. With widely varying plasma samples, the values showed a high degree correlation and good agreement. The intraassay variation was 3% which is in the acceptable range. The plasma samples could be analysed within 4 weeks and the reagents, were found to be stable, unlike some batches of trifluoro acetic acid (TFA).     

Proceedings: A method for preservation of bacteria and bacteriophages by drying in vacuo

An efficient and practical method was established to preserve bacterial strains and bacteriophages. The method is characterized by drying without freezing and by use of a cotton wool plug (nonabsorbent) to prevent contamination. Drying conditions were examined by measuring temperature, vacuum, and residual moisture of the samples. From the measurement, it was found that the cotton wool plug acts as a buffer and a desiccant. Thus, the specimens reached optimal conditions during storage. Another point of advantage is that the temperature of the specimen during the drying procedure was 2–5 °C; therefore, the evaporation of the water is rapid and the time of completion is shorter than that during lyophilization.     

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.     

Application of Flow Microcalorimetry to Analytical Problems: the Preparation, Storage and Assay of Frozen Inocula of Saccharomyces cerevisiae

A simple procedure for the freezing of large batches of yeast inocula (up to 150 samples) for storage at liquid nitrogen temperatures with the retention of high viability after thawing is described. The recovered frozen cells were examined using a flow microcalorimetric procedure which enables a rapid determination of viability. The application of such inocula to antibiotic assays and growth studies is discussed.     

玉米蛋白粉DNA提取方法比较研究

饲料样本本身的复杂性给进出口产品转基因（genetically modified,GM）成分的检测工作带来了巨大的压力和挑战。玉米蛋白粉主要包含蛋白质、淀粉和脂类等成分,从中提取高品质的DNA比较困难,而高品质的DNA是转基因检测研究的关键,能够大大降低进出口检验中的“假阴性”结果。采用市售主流品牌常用的7种试剂盒（Promega公司、Biotecon公司、天根生化科技有限公司、Invitrogen公司、Qiagen公司、TaKaRa公司）、CTAB法以及改良SDS?CTAB法提取玉米蛋白粉中的DNA。通过对玉米内源基因进行实时荧光PCR检测,发现改良SDS?CTAB法提取的玉米蛋白粉DNA质量明显优于其他方法,改良SDS?CTAB法内源基因zSSIIb的C_t值为28.14,较CTAB法提高了27%。研究建立的改良SDS?CTAB法在国内外尚属首次应用于饲料转基因产品中,并对7批次实验室送检样品进行转基因成分检测,成功检出2批次阳性样品。     

An efficient heuristic for scheduling batches of parts in a flexible flow system

Flexible manufacturing systems (FMSs) are a class of automated systems that can be used to improve productivity in batch manufacturing. Four stages of decision making have been defined for an FMS—the design, planning, scheduling, and control stages. This research focuses on the planning stage, and specifically in the area of scheduling batches of parts through the system. The literature to date on the FMS planning stage has mostly focused on the machine grouping, tool loading, and parttype selection problems. Our research carries the literature a step further by addressing the problem of scheduling batches of parts. Due to the use of serial-access material-handling systems in many FMSs, the batch-scheduling problem is modeled for a flexible flow system (FFS). This model explicitly accounts for setup times between batches that are dependent on their processing sequence. A heuristic procedure is developed for this batch-scheduling problem—the Maximum Savings (MS) heuristic. The MS heuristic is based upon the savings in time associated with a particular sequence and selecting the one with the maximum savings. It uses a two-phase method, with the savings being calculated in phase I, while a branch-and-bound procedure is employed to seek the best heuristic solution in phase II. Extensive computational results are provided for a wide variety of problems. The results show that the MS heuristic provides good-quality solutions.     

大批量测定植物(互花米草)叶面积的一种新方法

叶面积的准确测定是评价生态系统能流、碳流和水分利用的重要基础.目前常用的叶面积测定方法都有其局限性,有些方法快捷、准确,但仅适用于小批量测定;大多数方法很难在较短时间内准确进行大批量测定.本文以互花米草叶面积测定为例,探讨了以扫描仪和叶片干重为基础,结合PHOTOSHOP图像处理技术,大批量、快捷、准确测定植物叶面积的方法--扫描-干重法.结果表明:虚拟正方形叶片边长与叶子像素成幂函数曲线关系,公式为419.85x^1.9693,R≈1.00;用此公式验证扫描-干重法,相对误差仅为3.651%.用扫描-干重法测得的结果与方格法相比无显著差异(P=0.473),表明该方法是可行的.     

A Two-Stage Procedure for the Removal of Batch Effects in Microarray Studies

The presence of different batches is routinely observed in microarray studies and is well known that non-biological variability potentially confounding biological differences is commonly related to such batches. The removal of these undesired effects for a non-biased inference is often accomplished either with normalization methods that do not take into account all the available information, or with models that rely on strong parametric assumptions. We have developed a new method for the batch effects removal, named ber, which is based on a two-stage procedure for the estimation of location and scale parameters. Batch effects and biological differences are estimated using a regression approach and bagging, therefore mild distributional assumptions are required. We have compared ber with other commonly employed methods and we have shown that ber can bring to a higher power in detecting differentially expressed genes. The application of ber to a real microarray study led to interpretable biological results. The method is implemented in the R package ber, available through CRAN repository.     

Method for quantitative sampling of fungus gnat larvae in soilless growing media

A simple method is described for separating fungus gnat larvae from soilless growing media. Samples are first fractionated by water flotation with an inverted flask procedure and then the sediment is degassed under reduced air pressure and fractionated in magnesium sulfate (MgSO4) solution (density of 1.12 g cm(-3)). Fungus gnat larvae with only a small amount of contaminating debris are recovered from the surface of the MgSO4 solution for immediate counting or for preservation in alcohol. In evaluations of different commercial soilless growing media with a range of components, two repetitions of the water flotation step eliminated 20-40% of the dry weight of samples and virtually all of the perlite from further processing. Repeating both the water and MgSO4 flotations a third time only marginally improved the recovery of larval fungus gnats, Bradysia sp. nr. coprophila, added to pasteurized media. Extraction efficiency differed between instars and, to a lesser extent, between different types of media. Across three commercial soilless media tested, recovery was 24-33% for first, 68-85% for second, 85-95% for third, and 98-100% for fourth instars. Within combinations of media and instar, recovery was consistent. With this method, a 400-cm3 sample can be processed and be ready for counting in 1-1.5 h; samples can also be processed in batches or in assembly-line manner to process many samples per day. The method may also prove useful for quantitative recovery of shore fly larvae, thrips pupae, and other arthropods from soilless growing media.     

Plug flow cytometry: An automated coupling device for rapid sequential flow cytometric sample analysis.

BACKGROUND: The tools for high throughput flow cytometry have been limited in part because of the requirement that the samples must flow under pressure. We describe a simple system for sampling repetitively from an open vessel. METHODS: Under computer control, the sample is loaded into a sample loop in a reciprocating eight-way valve by the action of a syringe. When the valve position is switched, the plug of sample in the sample loop is transported to the flow cytometer by a pressure-driven fluid line. By coupling the plug-forming capability to a second multi-port valve, samples can be delivered sequentially from separate vessels. RESULTS: The valve is able to deliver samples at rates ranging up to about 9 samples per minute. Each plug of sample has uniform delivery characteristics with a reproducible coefficient of variation (CV). Even at the highest sampling rate, carryover between samples is limited. CONCLUSIONS: Plug-flow flow cytometry has the potential to automate the delivery of small samples from unpressurized sources at rates compatible with many screening and assay applications.     

A Two-Way Analysis of Covariance Model for Classification of Stability Data

This paper proposes a procedure for testing and classifying data with multiple factors. A two-way analysis of covariance is used to classify the differences among the batches as well as another factor such as package type and/or product strength. In the test procedure, slopes and intercepts of the main effects are tested using a combination of simultaneous and sequential F-tests. Based on the test procedure results, the data are classified into one of four different groups. For each group, shelf life can be calculated accordingly. We examine if the procedure produces satisfactory control of the probability of a Type I error and the power of detecting the difference of degradation rates and intercepts for different nominal levels. The method is evaluated with a Monte Carlo simulation study. The proposed procedure is compared with the current FDA procedure using real data.     

A modified procedure for the large scale preparation of tRNA from E. coli

A procedure for the preparation of about 50 g batches of tRNA from 25 kg E. coli W is described. The method involves phenolic extraction of the cells, batch absorption of the tRNA on DEAE-cellulose, washing the DEAE-cellulose and packing it into a column, elution of the tRNA from the column and precipitation of the tRNA with ethanol. The method is less time and labor consuming than the methods described in the literature and can be carried out with relatively simple equipment.     

Antibiotic Resistance Patterns of Enterococci and Occurrence of Vancomycin-Resistant Enterococci in Raw Minced Beef and Pork in Germany

The food chain, especially raw minced meat, is thought to be responsible for an increase in the incidence of vancomycin-resistant enterococci (VRE) in human nosocomial infections. Therefore, 555 samples from 115 batches of minced beef and pork from a European Union-licensed meat-processing plant were screened for the occurrence of VRE. The processed meat came from 45 different slaughterhouses in Germany. Enterococci were isolated directly from Enterococcosel selective agar plates and also from Enterococcosel selective agar plates supplemented with 32 mg of vancomycin per liter. In addition, peptone broth was used in a preenrichment procedure, and samples were subsequently plated onto Enterococcosel agar containing vancomycin. To determine resistance, 209 isolates from 275 samples were tested with the glycopeptides vancomycin, teicoplanin, and avoparcin and 19 other antimicrobial substances by using a broth microdilution test. When the direct method was used, VRE were found in 3 of 555 samples (0.5%) at a concentration of 1.0 log CFU/g of minced meat. When the preenrichment procedure was used, 8% of the samples were VRE positive. Our findings indicate that there is a low incidence of VRE in minced meat in Germany. In addition, the resistance patterns of the VRE isolates obtained were different from the resistance patterns of clinical isolates. A connection between the occurrence of VRE in minced meat and nosocomial infections could not be demonstrated on the basis of our findings.     

SIMSI-Transfer: Software-Assisted Reduction of Missing Values in Phosphoproteomic and Proteomic Isobaric Labeling Data Using Tandem Mass Spectrum Clustering

Isobaric stable isotope labeling techniques such as tandem mass tags (TMTs) have become popular in proteomics because they enable the relative quantification of proteins with high precision from up to 18 samples in a single experiment. While missing values in peptide quantification are rare in a single TMT experiment, they rapidly increase when combining multiple TMT experiments. As the field moves toward analyzing ever higher numbers of samples, tools that reduce missing values also become more important for analyzing TMT datasets. To this end, we developed SIMSI-Transfer (Similarity-based Isobaric Mass Spectra 2 [MS2] Identification Transfer), a software tool that extends our previously developed software MaRaCluster (© Matthew The) by clustering similar tandem MS2 from multiple TMT experiments. SIMSI-Transfer is based on the assumption that similarity-clustered MS2 spectra represent the same peptide. Therefore, peptide identifications made by database searching in one TMT batch can be transferred to another TMT batch in which the same peptide was fragmented but not identified. To assess the validity of this approach, we tested SIMSI-Transfer on masked search engine identification results and recovered >80% of the masked identifications while controlling errors in the transfer procedure to below 1% false discovery rate. Applying SIMSI-Transfer to six published full proteome and phosphoproteome datasets from the Clinical Proteomic Tumor Analysis Consortium led to an increase of 26 to 45% of identified MS2 spectra with TMT quantifications. This significantly decreased the number of missing values across batches and, in turn, increased the number of peptides and proteins identified in all TMT batches by 43 to 56% and 13 to 16%, respectively.     

Same-day batch measurement of glycine betaine, carnitine, and other betaines in biological material.

Glycine betaine, carnitine, carnitine esters, butyrobetaine, and proline betaine (stachydrine) concentrations in biological materials can be reliably measured in 100-microliters samples, with a detection limit below 1 mumol/liter. The procedure is suitable for batches of more than 30 specimens and it is possible to obtain a single result within 2 h. The betaines are extracted into an acetonitrile:methanol mixture, dried with anhydrous disodium hydrogen phosphate containing argentous oxide. The 4-bromophenacyl ester derivatives are formed using 4-bromophenacyl triflate as reagent, in the presence of solid magnesium oxide as base. The derivatives are separated by high-performance chromatography on a silica column, in a mixed partition and ion-exchange mode.     

Factors Affecting Triple Staining of Human Sperm

We have previously described a triple stain for evaluating normal acrosome reactions of human sperm. This procedure uses trypan blue to distinguish live and dead sperm, Bismarck brown to stain the sperm's postacrosomal region, and rose Bengal to stain the sperm's acrosome. We have recently found that batches of rose Bengal vary significantly in their ability to produce good staining of the acrosome in this procedure. This appears to be due to variations in the intrinsic pH of rose Bengal solutions and the presence of nondye contaminants in the stain. In this study, we have evaluated acrosomal staining using 6 batches of rose Bengal and report a method for achieving uniform staining quality with each batch. Solutions of rose Bengal (0.8%) are made up in 0.1 M Tris HC1 (pH 2.3) buffer and adjusted to pH 5.3 if necessary. For most batches of rose Bengal this promotes precipitation of some of the dye and an unidentified contaminating crystal. The precipitate is removed by centrifugation, and the supernatants have been found to give good to excellent staining of the acrosomes for all batches tested. Solutions of both rose Bengal and Bismarck brown are stable for at least 5 days but their pH values should be monitored daily and adjusted to 5.3 and 1.8 respectively if drifting occurs. We have also observed some variation in the intensity of rose Bengal staining of the acrosome from donor to donor and recommend that staining times in rose Bengal be adjusted for each donor.     

Factors affecting triple staining of human sperm

We have previously described a triple stain for evaluating normal acrosome reactions of human sperm. This procedure uses trypan blue to distinguish live and dead sperm, Bismarck brown to stain the sperm's postacrosomal region, and rose Bengal to stain the sperm's acrosome. We have recently found that batches of rose Bengal vary significantly in their ability to produce good staining of the acrosome in this procedure. This appears to be due to variations in the intrinsic pH of rose Bengal solutions and the presence of nondye contaminants in the stain. In this study, we have evaluated acrosomal staining using 6 batches of rose Bengal and report a method for achieving uniform staining quality with each batch. Solutions of rose Bengal (0.8%) are made up in 0.1 M Tris HCl (pH 2.3) buffer and adjusted to pH 5.3 if necessary. For most batches of rose Bengal this promotes precipitation of some of the dye and an unidentified contaminating crystal. The precipitate is removed by centrifugation, and the supernatants have been found to give good to excellent staining of the acrosomes for all batches tested. Solutions of both rose Bengal and Bismarck brown are stable for at least 5 days but their pH values should be monitored daily and adjusted to 5.3 and 1.8 respectively if drifting occurs. We have also observed some variation in the intensity of rose Bengal staining of the acrosome from donor to donor and recommend that staining times in rose Bengal be adjusted for each donor.     

Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production.

A protocol to test foetal calf serum (FCS) for contamination with bovine viral diarrhoea virus (BVDV) is described. Following this protocol, which combines cell culture methods and detection of pestivirus RNA, seven batches of FCS were tested. Infectious BVDV was detected in four of those batches. One of the remaining batches contained a relatively high number of non-infectious BVDV particles. A sample of this batch was formulated with aluminium hydroxide and aluminium phosphate as adjuvant into an experimental vaccine preparation. This product was injected twice into BVDV seronegative cattle with a 4 week interval. Blood samples taken 4 weeks after the second application were negative for BVDV specific antibodies. Our data stress that detection of BVDV RNA is not sufficient for a complete risk assessment on FCS. Discrimination between infectious and non-infectious BVDV is essential. This can only be achieved by cell culture methods.     

Exposure of in vitro-produced bovine embryos to foot-and-mouth disease virus

The aim of this study was to investigate whether foot and mouth disease virus (FMDV) interacts with in vitro produced (IVP) bovine embryos. One milliliter of a suspension of FMDV (2 x 10(7) TCID50/mL) was added to several batches of these embryos 7 d after in vitro fertilization, by which time they had either developed to the morula/blastocyst stage (n = 256) or degenerated (n = 260). Six experiments were performed in which developed or degenerated batches of embryos were incubated with FMDV for periods of 1 h (3), 2 h (2) or 4h (1). After this, the embryos were washed 10 times according to the International Embryo Transfer Society (IETS), then pooled and ground up to form a suspension, and assayed on cell cultures for FMDV. The cell cultures were observed daily for cytopathic effects for 3 d post exposure. In addition to the cell culture method, the polymerase chain reaction (PCR) technique was used to assay for the presence of the virus in the washing fluids. Assays for FMDV were also conducted on the first and second wash and on the pooled sample constituting the eight, ninth and tenth wash. With the exception of the second wash from a batch of embryos exposed to FMDV for 2 h, all samples of the first and second wash produced FMDV cytopathic effects, but none occurred with the pooled samples of the 8th, 9th and 10th wash. FMDV was also isolated from all but 1 of the batches of embryos after 1 h of incubation, from 1 of 4 batches after 2 h of incubation and from all batches after 4 h incubation. By contrast, the presence of virus could not be demonstrated by PCR based on the technique used here. These results show that 7 d old IVP bovine embryos can retain FMDV after washing, unlike in vivo-derived embryos, which do not appear to carry risks of FMDV transmission when washed according to IETS recommendations. Stricter controls are, therefore, necessary when using IVP embryos from cattle in a non-FMD-free zone in domestic or international trade.     

Methanol plug assisted sweeping-micellar electrokinetic chromatography for the determination of dopamine in urine by violet light emitting diode-induced fluorescence detection

The use and limitations of a methanol plug assisted sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method is described. Using naphthalene-2,3-dicarboxaldehyde (NDA)-labeled dopamine as a model compound, this new method was also used in the determination of dopamine in actual urine samples. An inexpensive violet light emitting diode (LED) was used for the light source, because this is suitable for fluorescence excitation. The number of theoretical plates of the analyte was determined to be approximately 1 x 10(5) and approximately 2 x 10(5) by means of MEKC and sweeping-MEKC and this was improved to approximately 1 x 10(6) when the methanol plug assisted mode was applied. In addition, the detection limit of NDA-labeled dopamine was determined to be 9.1 x 10(-7) and 1.2 x 10(-8)M by means of MEKC and sweeping-MEKC and this was improved to 4.7 x 10(-9)M when the methanol plug assisted sweeping-MEKC mode was applied.