A less laborious approach to the high-throughput production of recombinant proteins in Escherichia coli using 2-liter plastic bottles

Contemporary approaches to biology often call for the high-throughput production of large amounts of numerous proteins for structural or functional studies. Even with the highly efficient protein expression systems developed in Escherichia coli, production of these proteins is laborious and time-consuming. We have simplified established protocols by the use of disposable culture vessels: common 2-liter polyethylene terephthalate beverage bottles. The bottles are inexpensive, fit conveniently in commonly available flask holders, and, because they are notched, provide sufficient aeration to support the growth of high-density cultures. The use of antibiotics and freshly prepared media alleviates the need for sterilization of media and significantly reduces the labor involved. Uninoculated controls exhibited no growth during the time required for protein expression in experimental cultures. The yield, solubility, activity, and pattern of crystallization of proteins expressed in bottles were comparable to those obtained under conventional culture conditions. After use, the bottles are discarded, reducing the risk of cross-contamination of subsequent cultures. The approach appears to be suitable for high-throughput production of proteins for structural or functional studies.     

Instability of nitroglycerin tablets

Nitroglycerin is a volatile substance which evaporates from tablets if strict precautions are not taken. The tablets kept in small, amber, tightly capped glass bottles in a refrigerator maintain their potency for three to five months if bottles are opened once a week. After five months the unused tablets should be discarded. Tablets kept in a pill box and carried with the patient will deteriorate in a week and should be discarded thereafter. Cotton, plastic or paper stuffed in the bottle and frequent opening will reduce the potency of NG tablets considerably. Physicians, pharmacists and drug manufacturers should supply full instructions to patients on how to prevent rapid deterioration of their supply.     

Contamination of peritoneal dialysis fluid by filamentous fungi

Peritonitis is a frequent complication in peritoneal dialysis. It may be caused by contamination of the dialysis tubing or by extension of the catheter exit site. Gram-positive bacteria are the most common organism, accounting for 60% of all documented cases of continuous ambulatorial peritonitis dialysis. Fungi are isolated from to 1-15% of cases. Forty-nine out of 490 bottles containing fluid for peritoneal dialysis were randomly selected for microbiological analysis in S?o Paulo, Brazil. In this report the contamination of peritoneal dialysis fluid by Chaetomium globosum and Chrysonilia sitophila is reported.     

一次性氧气湿化瓶与重复使用湿化瓶应用的对比观察

目的：检测一次性氧气湿化瓶与重复使用湿化瓶用后细菌污染的程度和患者对噪音感受的舒适程度,对比两者的成本效益。方法：随机选出心内科病房内持续吸氧时间超过10天的患者100名,以随机分组的方法分出A组50名、B组50名。A组使用一次性氧气湿化瓶,B组使用重复使用的氧气湿化瓶。按照《医疗卫生机构消毒技术规范》进行采样后送微生物检验室进行病原学检验;同时对两组患者进行噪音感受舒适度的调查;根据使用的氧气湿化瓶成本费用、氧气湿化用灭菌注射用水的价格、含氯消毒剂健之素的费用计算成本。结果：50只一次性氧气湿化瓶使用时间120小时（5天）,微生物学检测未发现致病菌,成本费用9-3元／日,患者噪音感受舒适度满意度调查结果为100％;重复使用氧气湿化瓶使用24小时染茵率28％,成本费用9．6元／日,患者噪音感受舒适度满意度调查结果为60％。结论：使用合格的一次性氧气湿化瓶,在患者费用不会增加的前提下,减少了医院感染机会,增加了患者的舒适度,值得推广使用。     

MaxAlign: maximizing usable data in an alignment

Background  

The presence of gaps in an alignment of nucleotide or protein sequences is often an inconvenience for bioinformatical studies. In phylogenetic and other analyses, for instance, gapped columns are often discarded entirely from the alignment.     

Changes in Bacterial Numbers and Leucine Assimilation during Estimations of Microbial Respiratory Rates in Seawater by the Precision Winkler Method

During incubation of seawater in bottles, the decrease in dissolved oxygen is often nonlinear over time scales frequently used to measure respiration. Numbers of bacteria always increase, and rates of assimilation of dissolved leucine often increase exponentially. This suggests that sample handling disrupts the previously existing food web, leading to shifts of trophic state and unbalanced growth. Potential errors in measuring respiratory rate can be minimized by documenting these variables.     

No difference in colony formation of Scenedesmus obliquus exposed to lakes with different nutrient levels

The algal genus Scenedesmus is famous for its highly phenotypic plasticity in response to various environmental factors. In laboratory, axenic cultures of Scenedesmus often fail to form colonies and remain in unicellular morph. To examine whether unicellular Scenedesmus can form colonies after exposure to natural lakes, dialysis bags and plastic bottles which contained the precultured Scenedesmus obliquus were exposed in two lakes with different nutrient levels for four weeks. Results showed S. obliquus grew well in dialysis bags but not in plastic bottles; S. obliquus can form colonies when incubated in dialysis bags which allow exchange with the aquatic environment of the substances, regardless of the nutrient levels of the two lakes. However, no colonies were observed in S. obliquus incubated in plastic bottles exposed in both lakes. This suggested that active growth and zooplankton infochemicals contributed together to the colony formation of S. obliquus exposed in situ environment.     

A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. We examined the process of cell cycle exit in Drosophila wings and eyes and discovered that cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation.     

Computation of temperature-dependent dissociation rates of metastable protein–ligand complexes

Molecular simulations are often used to analyse the stability of protein–ligand complexes. The stability can be characterised by exit rates or using the exit time approach, i.e. by computing the expected holding time of the complex before its dissociation. However determining exit rates by straightforward molecular dynamics methods can be challenging for stochastic processes in which the exit event occurs very rarely. Finding a low variance procedure for collecting rare event statistics is still an open problem. In this work we discuss a novel method for computing exit rates which uses results of Robust Perron Cluster Analysis (PCCA+). This clustering method gives the possibility to define a fuzzy set by a membership function, which provides additional information of the kind ‘the process is being about to leave the set’. Thus, the derived approach is not based on the exit event occurrence and, therefore, is also applicable in case of rare events. The novel method can be used to analyse the temperature effect of protein–ligand systems through the differences in exit rates, and, thus, open up new drug design strategies and therapeutic applications.     

The Microflora of Hand Washed Milk Bottles

S ummary . The examination of a series of 713 milk bottles cleansed by hand washing at producer-retailer farm dairies showed that though nearly 60% attained satisfactory bacteriological standards, about 30% gave high colony counts (>600/bottle), while coli-aerogenes organisms were found in 17% and milk spoilage organisms in 25% of them. The microflora of efficiently cleansed bottles, with colony counts of <200/bottle, was dominated by micrococci and aerobic sporeforming rods. Only 3.7% of the 259 cultures from these bottles gave acid reactions in litmus milk in 72 h at 22°. Inefficiently cleansed bottles, with colony counts of >600/bottle, had quite a different type of microflora which was usually dominated by Gram negative rods (achromobacteria, nonfluorescent pseudomonads and flavobacteria). A much higher proportion (19%) of the 393 cultures from these bottles gave acid reactions in litmus milk.     

Bacterial Contamination of a Phenolic Disinfectant

Twenty ward stock bottles of aqueous 1% Printol were examined and 17 were found to be contaminated with Alcaligenes faecalis. Organisms were present in dead space behind the plastic liners of the bottle caps, where they could have survived washing. A. faecalis was also isolated from 31 out of 34 1% Printol solutions in use in the hospital. Pseudomonas aeruginosa was grown from two samples of 1% Printol in one ward, but not from stock bottles.The minimal bactericidal concentration (M.B.C.) in aqueous solution of Hycolin, Sudol, and Stericol for A. faecalis and Ps. aeruginosa was 1 in 320. The M.B.C. of Printol for both organisms was 1 in 80. The activity of all four disinfectants was reduced in the presence of large amounts of organic matter. Sudol was the least affected. Polyethylene, of which stock bottles were made, did not reduce the activity of the disinfectants. It is suggested that, ideally, stock bottles of disinfectant diluted ready for use should be autoclaved before they are refilled.     

Sterilization by Means of Hydrochloric Acid Vapour

As heat sterilization of glass bottles often results in breakage and of most plastic containers in deformation, we investigated a rapid low-temperature sterilization method as an alternative. Vapour evolving from hydrochloric acid was chosen because it does not leave toxic residues which might contaminate food packed in treated containers. Vapour evolving from 0.25 ml of 31% (w/w) hydrochloric acid reduced the number of viable spores of aerobic and anaerobic bacteria in bottles (300 ml) by a factor of at least 2500 and that from 0.01 ml of 37% (w/w) hydrochloric acid reduced the number of mould spores by a factor of over 40 000 within 5 min at 20°C.     

Continuous recombinant bacterial fermentations utilizing selective flocculation and recycle.

Selective recycle has successfully been used to maintain an unstable plasmid-bearing bacterial strain as dominant in a continuous reactor, whereas the culture reverts to 100% segregant cells when selective recycle is not used. The plasmid-bearing strain is slower growing and flocculent; however, when the cells lose their plasmid, the resulting segregant cells are nonflocculent and grow at a faster rate due to their decreased metabolic burden. Both types of cells exit a chemostat and enter an inclined settler where the flocculent plasmid-bearing cells are separated from the nonflocculent segregant cells by differential sedimentation. The underflow from the cell separator, which is enriched with plasmid-bearing cells, is recycled back to the chemostat, while the segregant cells are withdrawn off the top of the settler and discarded. The experimental results agree well with selective recycle reactor theory. On the basis of the theory, a criterion is presented that has been shown to successfully predict whether or not a selective recycle reactor can maintain a plasmid-bearing strain.     

Orbital shaker technology for the cultivation of mammalian cells in suspension

For large-scale applications in biotechnology, cultivation of mammalian cells in suspension is an essential prerequisite. Typically, suspension cultures are grown in glass spinner flasks filled to less than 50% of the nominal volume. We propose a superior system for suspension cultures of mammalian cells based on orbital shaker technology. We found that "square-shaped" bottles (square bottles) provide an inexpensive but efficient means to grow HEK-293 EBNA and CHO-DG44 cells to high density. Cultures in agitated 1-L square bottles exceeded the performance of cultures in spinner flasks, reaching densities up to 7 x 10(6) cells/mL for HEK-293 EBNA cells and 5 x 10(6) cells/mL for CHO-DG44 cells in comparison to (2.5-4) x 10(6) cells/mL for cultures of the same cells grown in spinner flasks. For 1-L square bottles, optimal cell growth and viability were observed with a filling volume of 30-40% of the nominal volume and an agitation speed of 130 rpm at a rotational diameter of 2.5 cm. Transient reporter gene expression following gene delivery by calcium phosphate-DNA co-precipitation was the same or slightly better for HEK-293 EBNA cells grown in square bottles as compared to spinner flasks. Reductions in cost, simplified handling, and better performance in cell growth and viability make the agitated square bottle a new and very promising tool for the cultivation of mammalian cells in suspension.     

The effects of certain antibiotics on biogas production in the anaerobic digestion of pig waste slurry

Antibiotics commonly used in the treatment of pigs - amoxicillin trihydrate, oxytetracycline hydrochloride and thiamphenicol were added at different concentrations to aliquots of pig waste slurry plus anaerobic sludge in serum bottles. The biogas production and methane concentration in the headspace were monitored to determine the effect of the antibiotics on the anaerobic process. With thiamphenicol significant differences in methane production were found for concentrations of 80 and 160 mg l(-1) slurry. Compared to the control, only minor differences in methane production were noted in the bottles to which amoxicillin (60 and 120 mg l(-1)) had been added. Methane production was about the same for the bottles with different oxytetracycline concentrations (125 and 250 mg l(-1)) and for the control.     

Clinical evaluation of polymerase chain reaction coupled with quantum dot fluorescence analysis in the identification of bacteria and yeasts in patients with suspected bloodstream infections

Bloodstream infections are serious and complex infectious diseases that often require a rapid diagnosis. Polymerase chain reaction coupled with quantum dot fluorescence analysis (PCR-QDFA) is a novel diagnostic technique. This study aimed to evaluate the diagnostic performance of PCR-QDFA for pathogen detection in patients with suspected bloodstream infections (BSIs). It evaluates 29 kinds of common pathogens (24 bacteria and 5 yeasts) from blood culture bottles. The results of PCR-QDFA identification and traditional microbial laboratory identification were compared, and the latter was used as the ‘gold standard’ to analyse the diagnostic performance of the PCR-QDFA. In total, 517 blood culture bottles were included in this study. The PCR-QDFA identified microorganisms in 368/422 (87.2%) samples with monomicrobial growth. For the pathogens on the PCR-QDFA list, the assay showed a higher sensitivity of 97.4% (368/378). When polymicrobial growth was analysed, the PCR-QDFA successfully detected 19/25 (76%) microorganisms on the PCR-QDFA list. In addition, 82/82 negative blood culture bottles also showed no pathogens by PCR-QDFA with a specificity of 100%. In conclusion, the PCR-QDFA assay could identify a majority of the common pathogens encountered in clinical practice, showing excellent diagnostic performance for pathogen detection in patients with suspected BSIs.     

Spinal nerves and their bearing on,salamander phylogeny

Examination of the vertebral columns of representatives of all families of salamanders revealed that, in contrast to the condition found in most other vertebrates, salamander spinal nerves often pass through foramina in the vertebrae. Two kinds of spinal nerve foramina were found: those in the anterior halves of vertebrae, and those in the posterior halves. In addition, many salamanders retain intervertebral nerves. However, within each family or, in a few cases, subfamily there is a characteristic pattern of spinal nerve-vertebral relationships. The first spinal nerve of all salamanders exits through a foramen in the anterior half of the atlas. All more posterior nerves are intervertebral in the families Cryptobranchidae, Hynobiidae and Proteidae. The posterior caudal nerves exit through the posterior halves of the caudal vertebrae in the family Amphiumidae, while in the subfamilies Dicamptodontinae and Rhyacotritoninae all post-sacral nerves exit through the posterior halves of the vertebrae. All but the first three nerves exit through posterior foramina in the family Plethodontidae and the subfamily Ambystomatinae, while all but the first two nerves pass through posterior foramina in the families Salamandridae and Sirenidae. Several fossil salamanders were also examined. These showed that the amphiumid and dicamptodontine-rhyacotritonine nerve patterns had evolved by the Late Cretaceous, and the sirenid pattern had probably evolved by that time. Other Cretaceous genera associated with the Ambystomatoidea still possessed the primitive intervertebral pattern. Using spinal nerve patterns and several other previously described morphological characters, a new hypothesis of the phylogeny of recent and fossil salamanders is presented and compared to earlier proposed phylogenies of the group. A new classification of salamander families is presented.     

Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Deltalte1 mutant and a Deltaras1 Deltaras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.     

Assessing gustatory detection capabilities using preference procedures

Two-bottle preference tests have often been used to make inferencesabout gustatory thresholds. The validity of such inferencesdepends on the extent to which taste differences produce differentialfluid consumptions from the bottles. If equal amounts are consumeddespite perceived gustatory dissimilarity, inaccurate thresholdestimates result. Modifications of common preference-test procedures,interacting with tastant properties and genetic variation, disclosedinstances of such non-discrimination among inbred strains ofmice. With an hedonically neutral to mildly unpalatable tastant(sodium cyclamate), taste-aversion conditioning yielded lowerthresholds. With a palatable tastant (maltose), testing withoutprior conditioning usually indicated lower thresholds. Descendingconcentration series produced lower thresholds than ascendingconcentration series, for both palatable (sucrose) and neutral(cyclamate) tastants. Previous experience with the tastantselevated preference scores, yielding lower thresholds with palatabletastants (sucrose, maltose) and higher thresholds with an unpalatabletastant (sucrose octaacetate). Within-strain discrepancies betweenthresholds indicated by the different methods were often large(2–3 log molar concentration steps). Amongstrain differencesof comparable magnitude, both in preferences and threshold estimates,were found for sucrose, maltose and cyclamate.