Quality assessment and analysis of Biogen Idec compound library

A subset of the compound repository for lead identification at Biogen Idec was characterized for its chemical stability over a 3-year period. Compounds were stored at 4 degrees C as 10 mM DMSO stocks, and a small subset of compounds was stored as lyophilized dry films. Compound integrity of 470 discrete compounds (Compound Set I) and 1917 combinatorial chemistry-derived compounds (Compound Set II) was evaluated by liquid chromatography/mass spectrometry from the time of acquisition into the library collection and after 3 years of storage. Loss of compound integrity over the 3 years of storage was observed across the 2 subsets tested. Of Compound Set I, 63% of samples retained > 80% purity, whereas 57% of samples from Compound Set II had purity greater than 60%. The stability of the lyophilized samples was superior to the samples stored as DMSO solution. Although storage at 4 degrees C as DMSO solution was adequate for the majority of compounds, the authors observed and quantified the level of degradation within the compound collection. Their study provides general insight into compound storage and selection of library subsets for future lead identification activities.     

The influence of storage conditions of tuna viscera before fermentation on the chemical, physical and microbiological changes in fish sauce during fermentation

Effect of storage condition of tuna viscera on chemical, physical and microbiological changes of its sauce were monitored. Results based on microbial counts, protein degradation products, total volatile base (TVB), and trimethylamine (TMA) contents, showed that tuna viscera stored at room temperature underwent more deterioration than that kept in ice, especially with increasing storage time. As a result, fish sauce obtained from tuna viscera stored at room temperature for a longer time rendered the greater amino nitrogen, TVB, TMA contents as well as browning intensity. However, storage conditions had no marked effect on overall changes in chemical, physical and microbiological characteristics of sauce generated during fermentation. Additionally, fish sauce produced from tuna viscera kept at room temperature comprised lower histamine content than that prepared from fresh or ice-stored viscera. Therefore, tuna viscera stored at room temperature for up to 8h could be used for the production of fish sauce with no detrimental effect on the quality.     

Seed storage of Brazilian cacti species in different threat categories

Cactaceae is considered the fifth most endangered taxonomic group. In light of this, the aim of this study was to evaluate the efficiency of different low‐temperature storage techniques in maintaining the viability of seeds of cacti in different threat categories. Seeds of six cacti taxa were stored in a cold chamber (8°C), a freezer (?5°C), in liquid nitrogen (?196°C) and at room temperature (25–27°C) for a period of 0, 1, 3, 6, 9 and 13 months. At each evaluation interval we removed a seed sample for each taxon studied, which was distributed into four repetitions of 25 seeds maintained at room temperature under 12‐h light/dark photoperiods. We evaluated the germinability, mean germination time and synchronization index. Most of the studied taxa presented germinability of above 50%, which was influenced by time and by storage temperatures. Also, most taxa stored at room temperature presented a significant reduction in germinability, whereas almost all taxa showed maintenance of the seed viability when stored in a cold chamber, a freezer or liquid nitrogen. This response can be justified by the reduction of the seed metabolism and the degradation of the reserve compounds of the seeds while at lower temperatures. Our results indicate that storage at low temperatures is an effective method for the conservation of cacti seeds and can be used for the formation of artificial seed banks of threatened cacti species.     

Investigation of 3 industry-wide applied storage conditions for compound libraries

The appropriate storage conditions for a compound file are a crucial factor for the success of drug discovery projects. In this study, 778 highly diverse compounds dissolved in 100% DMSO were stored under 3 industry-wide accepted storage conditions, and the compound integrity was monitored for a period of 6 months. The storage conditions selected were (1) under argon at +15 degrees C, (2) under argon at -20 degrees C, and (3) under ambient atmosphere at -20 degrees C. Each sample was assessed every 4 weeks by liquid chromatography coupled to mass spectrometry (LC/MS). Based on the resulting experimental data, a statistical projection of compound integrity over a period of 4 years for each of the 3 storage conditions was generated applying a linear mixed-effects model. A moderate loss of compound integrity of 12% was calculated for storage at -20 degrees C under argon, a loss of 21% for storage at -20 degrees C under ambient atmosphere, and a strong decrease of 58% for storage at +15 degrees C under argon over this period. The initial purity of the compounds does also influence the rate of compound degradation. Compounds with an initial purity of 50% to 75% degraded faster than compounds with an initial purity of more than 75%. The results of the study enable the prediction of the point in time, when the purity of a compound population falls below a predefined threshold that would trigger the resolubilization or retirement of the compound population represented by the analyzed samples.     

The effect of room-temperature storage on the stability of compounds in DMSO

The stability of approximately 7200 compounds stored as 20-mM DMSO solutions under ambient conditions was monitored for 1 year. Compound integrity was measured by flow injection analysis using positive and negative electrospray ionization mass spectrometry. Each sample was assessed at the beginning of the study, after 12 months of storage, and at a randomized time point between the initial and final time points of the study. The relationship between length of storage and the probability of observing the compound was described by a repeated-measures logistic regression model. The probability of observing the compound was 92% after 3 months of storage at room temperature, 83% after 6 months, and 52% after 1 year in DMSO. An acceptable limit for compound loss and corresponding maximum storage time for samples in DMSO can be determined based on these results.     

Characterization of the Fecal Microbiota Using High-Throughput Sequencing Reveals a Stable Microbial Community during Storage

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at −80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or −80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or −80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.     

不同干燥方式、存储温度对羊栖菜中岩藻黄素稳定性的影响

为探究不同干燥方式、不同存储温度和存储时间对羊栖菜中岩藻黄素稳定性的影响,本研究考察了三种不同干燥方式,包括自然阴干、低温烘干和冷冻干燥,对羊栖菜中岩藻黄素稳定性的影响。结果表明采用冷冻干燥所得羊栖菜,含水量低,岩藻黄素含量高,达到699.2μg/g,岩藻黄素保留率为94.7%,远高于自然阴干及低温烘干。因此,冷冻干燥是最佳干燥方法。考察了四个不同温度(-20、4、25、30℃)存储对冻干羊栖菜中岩藻黄素稳定性的影响,结果表明随着存储温度的升高,岩藻黄素的降解率明显增高,因此,-20℃为最佳储存温度。     

The effect of freeze/thaw cycles on the stability of compounds in DMSO

A diverse set of 320 compounds from the Procter & Gamble Pharmaceuticals organic compound repository was prepared as 20-mM DMSO solutions and stored at 4 degrees C under argon in pressurized canisters to simulate a low-humidity environment. The plates were subjected to 25 freeze/thaw cycles while being exposed to ambient atmospheric conditions after each thaw to simulate the time and manner by which compound plates are exposed to the atmosphere during typical liquid-handling and high-throughput screening processes. High-performance liquid chromatography-mass spectrometry with evaporative light-scattering detection was used to quantitate the amount of compound remaining after every 5th freeze/thaw cycle. Control plates were stored either at room temperature under argon or at 4 degrees C under argon without freeze/thaw cycling and were evaluated at the midpoint and the endpoint of the study. The study was conducted over a short time period (i.e., 7 weeks) to minimize the effect of compound degradation over time due to the exposure of the compounds to DMSO. The results from this study will be used to determine the maximum number of freeze/thaw cycles that can be achieved while maintaining acceptable compound integrity.     

Effect of Exposure Temperature on Over-All Efficiency of Autoradiographs and on Fogging of Emulsion

Systematic investigations of the influence of various temperatures of exposure on the autoradiographic efficiency and on the rate of fogging of Kodak AR 10 stripping film were carried out. The oxygen content and humidity of the exposed autoradiographs as well as the time of exposure were constant. Exposure of autoradiographs or storage of film at room temperature caused an increase of overall autoradiographic efficiency by approximately 40%, but although the rate of film fogging was also increased, it did not exceed the safe values of background. Both autoradiographic efficiency and rate of fogging were not altered significantly when a temperature of 4 C or -18 C for exposure was used. Therefore, room temperature of exposure is recommended for ordinary autoradiography. For a special autoradiographic technic when low temperature of exposure has to be used, the temperature -18 C may be safely applied without significant impairment of autoradiographic efficiency.     

Storage stability of Anagrapha falcifera nucleopolyhedrovirus in spray-dried formulations

A multiply embedded nucleopolyhedrovirus isolated from Anagrapha falcifera (Kirby) (AfMNPV) can lose insecticidal activity during months of dry storage in ambient room conditions. We tested the spray-dried AfMNPV formulations after storage for up to 1 year at room temperatures for insecticidal activity against neonate Trichoplusia ni (Hübner). Experimental formulations were made using combinations of corn flours, lignin, and sucrose, and were selected based on previous work which demonstrated that these formulations resisted solar degradation in field experiments. Twelve experimental formulations (organized in three groups of four formulations) compared the effect of (1) the ratio of formulation ingredients (lignin and corn flour) to virus concentration, (2) different sources of lignin, or (3) different corn flours and sugar. Based on a single-dose plant assay with these 12 formulations, none of the formulations lost significant activity due to the drying process, when compared with the unformulated wet AfMNPV. Samples of the 12 dried formulations were stored at room (22+/-3 degrees C) and refrigerated (4 degrees C) temperatures. Insecticidal activity (LC(50)) was determined with a dosage-response assay for neonates fed on treated cotton-leaf disks. After 6 (or 9) and 12 months storage, refrigerated samples maintained insecticidal activity better than corresponding samples stored at room temperatures with LC(50)s that averaged 2.0 x 10(6) polyhedral inclusion bodies per milliliter (pibs/ml) for refrigerated samples and 5.4 x 10(6) pibs/ml for samples stored at room temperatures. Compared with unformulated stock virus stored frozen, six formulations stored at room temperature and 10 formulations stored in the refrigerator did not lose significant insecticidal activity after 1 year based on overlapping 90% confidence intervals. Changing the ratio of virus to formulation ingredients did not provide a clear trend over the range of concentrations tested, and may be less important for shelf-life of virus activity compared with formulations made with different ingredients. Two of the four formulations made with different lignins were about 15 times less active after 1 year at room temperature compared with refrigerated samples, indicating that specific formulation ingredients can affect storage stability. Formulations that contained sugar generally maintained activity during storage better than formulations without sugar. Unformulated virus stock maintained insecticidal activity (ranged from 0.20 to 2.5 x 10(6) pibs/ml) better during storage than dried formulations with LC(50)s that ranged from 0.39 to 27 x 10(6) pibs/ml. Unformulated virus stock, which is essentially a suspension of virus occlusion bodies in homogenized insect cadavers, did not lose activity when stored at refrigerated or room temperature. We believe that stability of AfMNPV insecticidal activity during storage as dry formulations is related to the general composition of the formulation and that sugar may play a critical role in maintaining insecticidal activity.     

假酸浆种子发芽特性研究

在不同温度、不同贮藏时间和方法下,对来源于印度尼西亚的假酸浆种子发芽特性进行研究。结果表明:不同温度对假酸浆种子萌发的影响差异极显著,在15/25℃变温条件下种子发芽率最高,为84.3%,发芽快且整齐;假酸浆种子采收后立即播种其发芽率非常低,室温下贮藏6～9个月时在25℃恒温或15/25℃变温条件下均有较高发芽率,说明假酸浆种子有休眠性,通过延长贮藏时间能打破休眠,促进种子发芽;但室温贮藏15个月后,种子活力下降非常明显。低温冷藏在一定条件下能提高假酸浆种子萌发能力,能延长种子寿命。     

Paper-based archiving of mammalian and plant samples for RNA analysis

The ability to archive biological samples for subsequent nucleic acid analysis is essential for tissue specimens and forensic samples. FTA Card is a chemically treated filter paper designed for the collection and room temperature storage of biological samples for subsequent DNA analysis. Its usefulness for the preservation of biological samples for subsequent RNA analysis was tested. Here, we demonstrate that RNA in biological samples stored on FTA Cards is stable and can be used successfully for RT-PCR and northern blot analysis. RNA stability depends on the storage temperature and the type of biological specimen. RNA in mammalian cells stored on FTA Cards is stable for over one year at temperatures at or below -20 degrees C and for two to three months in samples stored at room temperature. For plant leaf, longer storage times (> 5 days) require temperatures at or below -70 degrees C following sample application. FTA Cards may constitute a method not only for convenient collection and storage of biological samples but also for rapid RT-PCR analysis of tissue and cell samples.     

不同贮藏温度下大别山五针松花粉活力的变化

大别山五针松( Pinus dabeshanensis C. Y. Cheng et Y. W. Law)为松科( Pinaceae)松属( Pinus Linn.)植物,自然种群数量极少,目前仅发现在安徽省岳西县大王沟海拔900~1300 m阴坡和半阴坡有相对集中的分布,种群规模计200余株,且多为成年个体,林下幼苗极少,自然更新困难,为中国特有珍稀树种之一[1]。大别山五针松常在每年3月份至4月份形成花芽,5月中下旬花粉成熟并散发,花粉具气囊,此时雌球花张开接受传粉,球果翌年9月成熟[2]。据作者近年的调查,大别山五针松种子败育率较高,且种子质量较低,这也是该种类濒危的重要原因之一。     

The effects of time and temperature on flow cytometry enumerated live Cryptosporidium parvum oocysts

AIMS: Recoveries of spiked standard suspensions are used to evaluate method performance. For many applications, gamma-irradiated Cryptosporidium oocysts are appropriate. In contrast, methods that determine viability, such as Cryptosporidium cell culture, require the use of live oocysts. Oocyst standards are usually prepared at a flow cytometry laboratory for use at another laboratory, and thus the samples are shipped. The goal of this study was to evaluate the shipping and storage stability of flow cytometry enumerated live oocysts over time at three temperatures: 4 degrees C, room temperature and 37 degrees C. METHODS AND RESULTS: Replicate samples containing 100 live C. parvum oocysts were prepared by flow cytometry and stored at 4 degrees C, room temperature and 37 degrees C. These samples were counted at various time points. Significant oocyst losses were observed after storage for 1 day at 37 degrees C, 7 days at room temperature and 21 days at 4 degrees C. CONCLUSIONS: Live C. parvum oocysts internal standards should be used within 10 days of preparation, and stored and shipped at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: When evaluating method performance with live oocysts, both the storage temperature and time are critical factors for obtaining reliable and accurate results.     

Effect of temperature and time of storage of dry soil samples on a chemical index of N availability

Summary The effect was studied of storage temperature on the index of available soil N wich uses U.V. absorbance of a 0.01M NaHCO₃ extract as an indicator. The U.V. absorbance was found to increase at a non-linear rate for four soils stored at temperatures of 50, 75, and 150°C. The change in extract absorbance due to extended soil storage at each of these temperatures was positively correlated to the percent organic matter, percent N, C/N value and concentration of humic substances in soils, but not to the extract absorbance prior to soil storage. These findings were not consistent with room temperature storage data which showed a linear increase in extract absorbance with soil storage time. The change in absorbance for the room temperature case was not related to any of the soil parameters mentioned above. Analysis of a soil stored at 105°C showed an increase in ninhydrin-detectable N, protein N and Kjeldahl N of the NaHCO₃ extract, while the apparent molecular weight distribution of extracted organic matter (as determined by gel filtration) showed only a slight change. As a comparison to the NaHCO₃ extract, a boiling CaCl₂ extract of the same soil was also analyzed; and the absorbance at 260 nm was found to increase in a curvilinear fashion with starage time at 75°C but to less of an extent than was noted with the NaHCO₃ extract. Nitrogen availability indexes based on the U.V. absorbance of these extracts, particularly those utilizing the NaHCO₃ extract, would be significantly affected by soil storage at elevated temperatures.Paper No. 6176 of the J. Ser. of the Pennsylvania Agric. Exp. Stn. Authorized for publication January 26, 1981.     

Patulin accumulation in apples by Penicillium expansum during postharvest stages

AIMS: The aim of this study was to assess the opportunities of Penicillium expansum to develop and produce patulin in apples during cold storage and in the steps prior to processing of apple products. METHODS AND RESULTS: Two lots of apples var. Golden with different ripeness degree were used. Half of each lot was fungicide treated. Apples were inoculated with P. expansum and stored at 1 degrees C for 6 weeks. The extent of lesions and patulin accumulation both at the end of cold storage and after 3 days at 20 degrees C were assessed. Short storage at 20 degrees C aimed to simulate the transport and storage steps at room temperature before processing. Lesion size significantly increased during the storage at 20 degrees C. An interaction between fungicide treatment and ripeness degree was found; efficiency of fungicide treatment was higher for ripe apples. Although lesions were evident after cold storage, no patulin was detected. Patulin was detected only when fruits were further stored at 20 degrees C. Neither ripeness degree nor fungicide treatment affected patulin accumulation. CONCLUSIONS: Cold storage periods of 6 weeks do not lead to patulin accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: Shortening preprocessing times at warm temperatures would result into a reduction in patulin content at initial steps of fruits entering the processing plants.     

Growth of enterotoxigenic Staphylococcus aureus in povi masima, a traditional Pacific island food

AIMS: To obtain preliminary data on the microbiology and hurdles to pathogen growth in the traditional Pacific Island food, povi masima, which is essentially beef brisket cured in brine. METHODS AND RESULTS: Six containers of povi masima were prepared and two were inoculated with five enterotoxigenic strains of Staphyloccocus aureus. The povi masima were divided into two lots each containing two uninoculated control and an inoculated container. Lot 1 was incubated at room temperature (20 degrees C) and lot 2 under refrigeration (4-5 degrees C) for up to 98 days. During storage, samples were removed and tested for aerobic plate count, coagulase-producing Staphylococci, Clostridium perfringens, staphylococcal enterotoxin and various chemical parameters of the food. Coagulase-producing Staphylococci and aerobic plate counts grew to high levels in both the inoculated and uninoculated lots stored at room temperature, but enterotoxin was only detected at one time point in these lots and this may represent a false positive result. The concentration of NaCl in the meat increased with time as concentrations equilibrated, and nitrite was rapidly lost in those lots stored at room temperature. Storage at 4-5 degrees C prevented proliferation of coagulase-producing Staphylococci. CONCLUSIONS: For safe curing and storage, this food should be kept under refrigeration as this prevented growth of staphylococci. Optimum storage would also be achieved with improved attempts to ensure equal distribution of NaCl prior to storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Under conditions traditionally used to cure and store this food, enterotoxigenic staphylococci can grow to numbers where toxigenesis might occur, especially during the early stages of curing where the salt has not diffused from the brine into the meat.     

Isolation of high-molecular-weight DNA from small samples of blood having nucleated erythrocytes,collected, transported,and stored at room temperature

Blood samples collected in the field for isolating DNA suitable for molecular analysis need special care in their storage and handling. In this article, we describe a simple method for the isolation of good-quality high-molecular-weight DNA that does not require low temperature conditions during collection, storage, and/or transportation of blood samples. This method involves smearing small aliquots of blood onto clean slides and air drying them at room temperature. The slides with blood smears can then be transported or stored at room temperature and still serve as a very good source of high-molecular-weight DNA. Genomic DNA from these samples can be extracted by organic phase separation (phenol-chloroform extraction) after lysis. The DNA thus obtained is of high quality and yields DNA fingerprints qualitatively similar to those prepared from corresponding control DNA isolated from frozen blood samples. Needing minimal facilities at field sites, the method is very convenient for conducting RFLP analysis of wild/field populations for demographic, behavioral, and ecologic studies.     

Long-term storage and viability of Ochlerotatus albifasciatus (Diptera: Culicidae)

The viability of Ochlerotatus albifasciatus (Macquart) eggs stored at room temperature and at 5 degrees C was studied over 31 months. After 12, 18 and 31 months of storage, eggs were acclimatized at 22 degrees C for ten days, and then inundated twice every seven days. The effect of the storage period on the percentage of hatching was analyzed by one way ANOVA. Differences on the hatching response between the first and second flooding were analyzed by paired t-test. Differences on the hatching response between the two storage conditions were analyzed by Mann-Whitney rank test. Results showed that (1) Oc. albifasciatus eggs were able to survive and hatch over 31 months; (2) the percent hatching of eggs stored at 5 degrees C was higher than that of eggs stored at room temperature; and (3) low temperatures and long periods without water favor installment hatching.     

Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures.

The long-term storage of pathogenic and nonpathogenic strains of both Naegleria and Acanthamoeba spp. were tested on Page amoeba saline agar slopes for 24 months at room temperature and for 8 months at -10, 4, 10, and 15 degrees C. Acanthamoeba strains showed better survival potential than Naegleria strains, particularly when they were stored at temperatures equal to or lower than room temperature.