Technical strategies to reduce the amount of "false significant" results in quantitative proteomics

When the p-value is set at <0.05 in statistical group comparisons, a 5% rate of "false significant" results is expected. In order to test the reliability of our 2-DE method, we loaded each of 24 gels with equal-sized samples (200 mug protein from pooled rat brain, pH 4-7, stained with ruthenium fluorescent stain for visualization) and statistically compared the first 12 gels with the last 12. In numerous experiments the rate of significant differences found far exceeded 5%. Several factors were identified as causing the following rates of false significant differences in spot intensities: (i) running samples in two different 2-DE runs (42%), (ii) running second dimension gels produced in two different gel casters (16%), (iii) normalizing the entire gel instead of separately normalizing several different gel zones (11%), (iv) using IPG strips from different packages (19%), (v) dividing the whole sample into subgroups during software analysis (9%). After controlling for all these factors, the rates of "false positive" results in our experiments were regularly reduced to approximately 5%. This is an indispensable prerequisite for avoiding too high a rate of false positive results in experiments in which different subgroups are compared statistically.     

Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension

It has been shown that a decrease in oxygen tension during cultivation of multipotent mesenchymal stromal cells (MMSCs) caused a short-term decline in the proportion of CD73⁺ cells in the population, with no effect on the number of cells expressing the other constitutive surface markers (CD90, CD105). The heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable under conditions close to tissue oxygen levels (5% O₂). At lower oxygen concentration, it is gradually reduced from the third to fourth passages. The increase in proliferative activity was not accompanied by increased telomerase gene expression, which indicated that no cell transformation had occurred. Global gene expression analysis of MMSC gene expression revealed changes in expression of cyclins (CCND2, PCNA), regulatory subunit cyclin-dependent kinase (CKS2), and an inhibitor of cyclin-dependent kinases 4 and 6 (CDKN2C) regulating the cell cycle, which probably facilitated the proliferative activity of cells at lower oxygen tension.     

A technique for regulating oxygen tension in a closed biological system

Summary A technique was developed for maintaining the desired oxygen level in biologically active closed systems. In this method, the chemical mechanism used for replenishing oxygen in the system is directly related to O₂ consumption and CO₂ production.     

Elicitation and abrupt termination of behaviorally significant catchlike tension in a primitive insect

Sustained steady contractural or catchlike tension (CT) occurs in the metathoracic extensor tibiae muscle of the primitive insect the weta (Orthoptera: Stenopelmatidae) during its characteristic leg-extension defense behavior or following leg-position conditioning. Similar action occurs occasionally in semi-intact preparations and is abruptly turned off by a single peripheral inhibitory impulse. These phenomena were reproduced routinely by first infusing saline containing 10^?8M (or stronger) octopamine into the muscle for 12 min, and then stimulating the slow excitatory motor neuron SETi with a brief burst. Direct stimulation of the dorsal unpaired median neuron, innervating the extensor tibiae (DUMETi) prior to SETi stimulation, also led to CT. Both octopamine and DUMETi markedly enhanced the tension developed in response to a burst of impulses in SETi.     

Experimental study of results of nerve suture under tension vs. nerve grafting.

Evoked electromyograms and myelinated fiber caliber histograms were used to compare the results of end-to-end nerve suture and nerve grafting, performed with an operating microscope. The best results were achieved after direct suture without tension. Direct suture under a tension of 25 grams produced results sometimes comparable the that of the zero tension group, but mostly quite inferior to those and even to the grafted group. The regeneration of axons in the nerve graft group was delayed more than in the sutured groups, but by the tenth month the number of regenerating fibers had increased with stable and comparatively good results. After a nerve suture under a tension of 25 grams, there is impairment of the blood flow at the suture site, and the results are not as good. In such situations I believe nerve grafting should be performed. This amount of tension also seems to be the limit which will allow accurate coaptation of a nerve by microsurgical techniques.     

Early results of closed partial menisectomy.

Thirty patients underwent partial meniscectomy through two puncture incisions by a closed technique under arthroscopic control. "Bucket-handle" fragments were removed from 17 knees, and flaps or tags from the remainder. The mean stay in hospital after operation was 1.3 days and the mean time for return to working fitness was 10.5 days. There were no serious complications. Further study of the long-term results of this technique is needed.     

Relationship between mechanical properties and collagen structure of closed and open wounds

Mechanical properties and collagen structure of excisional wounds left open are compared with wounds closed by clips. In both wound models, collagen fiber diameter increases with time post-wounding and is related to tensile strength. Clipped wounds show a higher ultimate tensile strength and tangent modulus compared with open wounds. In clipped wounds, newly deposited collagen appears as a biaxially oriented network as observed in normal skin. In open wounds a delay in the organization of the collagen network is observed and parallel wavy-shaped ribbons of collagen fibers are deposited. At long term, the high extensibility observed in open wounds may be due to the sliding of ribbons of collagen fibers past each other.     

Rupture of plasma membrane under tension

We present a study on the rupture behavior of single NIH 3T3 mouse fibroblasts under tension using micropipette aspiration. Membrane rupture was characterized by breaking and formation of an enclosed membrane linked to a tether at the cell apex. Three different rupture modes, namely: single break, initial multiple breaks, and continuous multiple breaks, were observed under similar loading condition. The measured mean tensile strengths of plasma membrane were 3.83 ± 1.94 and 3.98 ± 1.54mN/m for control cells and cells labeled with TubulinTracker, respectively. The tensile strength data was described by Weibull distribution. For the control cells, the Weibull modulus and characteristic strength were 1.86 and 4.40 mN/m, respectively; for cells labeled with TubulinTracker, the Weibull modulus and characteristic strength were 2.68 and 4.48 mN/m, respectively. Based on the experimental data, the estimated average transmembrane proteins-lipid cleavage strength was 2.64 ± 0.64 mN/m. From the random sampling of volume ratio of transmembrane proteins in cell membrane, we concluded that the Weibull characteristic of plasma membrane strength was likely to be originated from the variation in transmembrane proteins-lipid interactions.     

Long-term results of MISTI gold breast implants: a retrospective study

MISTI Gold breast implants (Bioplasty, St. Paul, Minn.) filled with polyvinylpyrrolidone-hydrogel were developed as a promising alternative to silicone-filled implants. Some studies have reported on the positive effects of the implant, such as improved radiolucency and biocompatibility of the gel; however, there are also reports that such implants increased in volume and were subject to capsular contracture in the human body, resulting in demands for their removal. The purpose of this retrospective study was to analyze the long-term results of a series of patients with MISTI Gold breast implants. Between 1991 and 1993, the authors inserted 83 MISTI Gold implants in 61 patients with an average age of 46 years (range, 16 to 69). The authors were able to follow up 48 patients with 71 MISTI Gold implants. The average follow-up was 68 months (range, 10 to 108 months). The retrospective study found that 59 percent of all MISTI Gold implants were removed after an average period of 4.14 years. The main reason for implant removal was an increase in volume of 38 percent, followed by capsular contracture in 14 percent of all 71 MISTI Gold implants. The average increase in volume of all removed MISTI Gold implants was 43 percent. Capsular contracture was graded as Baker I and II in 63 percent and as Baker III and IV in 37 percent. In conclusion, the authors believe that MISTI Gold implants do not fulfill the criteria of safe breast implants, and they agree with the December of 2000 opinion of the Medical Devices Agency of the Department of Health in London that the hydrogel-filled breast implants should not be used until more information about the filler material and its metabolic fate is available.     

Long-term results following velopharyngoplasty with a cranially based pharyngeal flap.

Between 1980 and 1989, 82 velopharyngoplasties have been carried out in the Department of Oral and Maxillofacial Surgery at the Medical University of Hannover. Speech results of 51 of these patients, including 39 patients with cleft lip and palate, could be followed up in the context of a clinical follow-up examination. Besides evaluation of speech results by two senior speech pathologists and two untrained listeners, a frequency analysis of the speech results with a sonograph was obtained. Nasal air loss was documented with a fogged-mirror test and computer aerometry. Whereas in 37 of 51 patients a normal or almost normal colloquial speech could be demonstrated, 30 of 39 patients with cleft lip and palate showed a normal or almost normal realization of the test sentences. Thirty of the 37 patients (81.08 percent) with normal or almost normal colloquial speech showed extensive mobility of the lateral pharyngeal wall. Symmetry of the velopharyngeal flaps seemed to have no influence on the speech result. With a fogged-mirror test, an average reduction of mirror fogging from 2.0 rings preoperatively to 0.9 rings postoperatively could be shown. In 31 patients, there was no longer any air loss postoperatively. Besides one rupture of a flap, two flaps had to be diminished in their lateral dimensions because of excessive size. We regard the cranially pedicled pharyngeal flap as an important operative procedure for improving speech results, especially in cleft lip and palate patients.     

Model of urethra under uniform circumferential tension

The urethra as seen on X-ray films may show alternate regions of constriction and distension. That these regions do not necessarily correspond to high and low tensions in the circumferential muscle sheath is shown by calculated stable configurations under uniform tension.     

Phase behavior of lipid bilayers under tension

Given the proposed importance of membrane tension in regulating cellular functions, we explore the effects of a finite surface tension on phase equilibrium using a molecular theory that captures the quantitative structure of the phase diagram of the tensionless DPPC/DOPC/Cholesterol lipid bilayer. We find that an increase in the surface tension decreases the temperature of the transition from liquid to gel in a pure DPPC system by ～1.0 K/(mN/m), and decreases the liquid-disordered to liquid-ordered transition at constant chemical potentials by approximately the same amount. Our results quantitatively isolate the role of tension in comparison to other thermodynamic factors, such as pressure, in determining the phase behavior of lipid bilayers.     

Minimum amount of penicillin prophylaxis required to control Streptococcus pyogenes epidemic in closed community.

The prophylaxis required to control an epidemic of Streptococcus pyogenes throat infection in a junior detention centre has been reported. In a further epidemic an attempt was made to determine the minimum amount of penicillin required to control the outbreak. Oral penicillin (0.5 g) given as a single daily dose for 10 days to all boys after entry proved effective. The added risk of relatively deprived adolescent boys developing rheumatic fever is stressed.     

Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system

The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.