Preparing nuclei from cells in monolayer cultures suitable for counting and for following synchronized cells through the cell cycle

A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.     

Succession of mycorrhizal fungi in stands of Pinus sylvestris in the Netherlands

The mycorrhizal mycoflora was investigated in 35 stands of Pinus sylvestris in three types of young (4-13 yr) and three of old (50-80 yr) stands in the Netherlands, differing in number of rotations and soil type. A plot of 1050 m² (30 m x 35 m) within each stand was searched for carpophores during the autumns of 1986 and 1987. 10 soil samples per plot were taken in October 1987 in order to assess the mycorrhizal status of the tree roots. The composition of mycorrhizal mycoflora in the different plots was subjected to TWINSPAN cluster analysis and Detrended Correspondence Analysis. Plot groupings generated by these analyses largely parallelled the stand types, indicating that each stand type has its own mycoflora. Differences in myco-floristic composition between stand types were parallelled by differences in the composition of green vegetation. The young stand types had 3.5–27 x more carpophores and 1.4–6.8 x more species than two of the old stand types One old stand type was intermediate. Considerable differences in species composition between the young stand types were observed. It is concluded that the succession of mycorrhizal fungi is not primarily influenced by ageing of the trees, but rather by changes in the soil. The results were compared with data on changes in the occurrence of fruiting species of mycorrhizal fungi in the Netherlands during this century. It appeared that species which have declined according to these data were more frequent in the young plots than in the old plots. However, these species are reported to be frequent in old stands of P. sylvestris in Estonia and Finland. It is argued that this difference is related to the high nitrogen deposition in the Netherlands.     

Finite element analysis of different loading conditions for implant-supported overdentures supported by conventional or mini implants

The effect of implants’ number on overdenture stability and stress distribution in edentulous mandible, implants and overdenture was numerically investigated for implant-supported overdentures. Three models were constructed. Overdentures were connected to implants by means of ball head abutments and rubber ring. In model 1, the overdenture was retained by two conventional implants; in model 2, by four conventional implants; and in model 3, by five mini implants. The overdenture was subjected to a symmetrical load at an angle of 20 degrees to the overdenture at the canine regions and vertically at the first molars. Four different loading conditions with two total forces (120, 300 N) were considered for the numerical analysis. The overdenture displacement was about 2.2 times higher when five mini implants were used rather than four conventional implants. The lowest stress in bone bed was observed with four conventional implants. Stresses in bone were reduced by 61% in model 2 and by 6% in model 3 in comparison to model 1. The highest stress was observed with five mini implants. Stresses in implants were reduced by 76% in model 2 and 89% increased in model 3 compared to model 1. The highest implant displacement was observed with five mini implants. Implant displacements were reduced by 29% in model 2, and increased by 273% in model 3 compared to model 1. Conventional implants proved better stability for overdenture than mini implants. Regardless the type and number of implants, the stress within the bone and implants are below the critical limits.     

CAT & MAUS: A novel system for true dynamic motion measurement of underlying bony structures with compensation for soft tissue movement

Optoelectronic motion capture systems are widely employed to measure the movement of human joints. However, there can be a significant discrepancy between the data obtained by a motion capture system (MCS) and the actual movement of underlying bony structures, which is attributed to soft tissue artefact. In this paper, a computer-aided tracking and motion analysis with ultrasound (CAT & MAUS) system with an augmented globally optimal registration algorithm is presented to dynamically track the underlying bony structure during movement. The augmented registration part of CAT & MAUS was validated with a high system accuracy of 80%. The Euclidean distance between the marker-based bony landmark and the bony landmark tracked by CAT & MAUS was calculated to quantify the measurement error of an MCS caused by soft tissue artefact during movement. The average Euclidean distance between the target bony landmark measured by each of the CAT & MAUS system and the MCS alone varied from 8.32 mm to 16.87 mm in gait. This indicates the discrepancy between the MCS measured bony landmark and the actual underlying bony landmark. Moreover, Procrustes analysis was applied to demonstrate that CAT & MAUS reduces the deformation of the body segment shape modeled by markers during motion. The augmented CAT & MAUS system shows its potential to dynamically detect and locate actual underlying bony landmarks, which reduces the MCS measurement error caused by soft tissue artefact during movement.     

Lower limb joint work and joint work contribution during downhill and uphill walking at different inclinations

Work performance and individual joint contribution to total work are important information for creating training protocols, but were not assessed so far for sloped walking. Therefore, the purpose of this study was to analyze lower limb joint work and joint contribution of the hip, knee and ankle to total lower limb work during sloped walking in a healthy population. Eighteen male participants (27.0 ± 4.7 yrs, 1.80 ± 0.05 m, 74.5 ± 8.2 kg) walked on an instrumented ramp at inclination angles of 0°, ±6°, ±12° and ±18° at 1.1 m/s. Kinematic and kinetic data were captured using a motion-capture system (Vicon) and two force plates (AMTI). Joint power curves, joint work (positive, negative, absolute) and each joint’s contribution to total lower limb work were analyzed throughout the stance phase using an ANOVA with repeated measures. With increasing inclination positive joint work increased for the ankle and hip joint and in total during uphill walking. Negative joint work increased for each joint and in total work during downhill walking. Absolute work was increased during both uphill (all joints) and downhill (ankle & knee) walking. Knee joint contribution to total negative and absolute work increased during downhill walking while hip and ankle contributions decreased. This study identified, that, when switching from level to a 6° and from 6° to a 12° inclination the gain of individual joint work is more pronounced compared to switching from 12° to an 18° inclination. The results might be used for training recommendations and specific training intervention with respect to sloped walking.     

The Concept of Additivity of Drug Combinations in the Case of Quantal Response

The concept of additivity of drug combinations is widely accepted in pharmacology and toxicology. Up to now, no general statistical methods to test that property are available. The present paper gives a mathematical formulation of additivity, a method to fit dose response surfaces under additivity assumption and a statistical test.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.