Centrifugation protocol for the NASA Artificial Gravity-Bed Rest Pilot Study

We have implemented a 41-day ground-based study to investigate the effects of daily artificial gravity loading on bed rest deconditioned human subjects. Each subject underwent 21 days of 6 degree head-down bed rest. Treatment subjects received 60 min daily doses of inertial mechanical loading (2.5 G at the feet decreasing to 1 G at the heart) produced by a short radius centrifuge. During rotation, the subject's cardiovascular responses were monitored via ECG, blood pressure and pulse oximetry, and subjective assessment of motion sickness and overall health were periodically requested. The subject's weight distribution at the feet was measured using a force plate, and lower leg muscle activity was monitored via surface electromyography. Control subjects were instrumented but did not receive any centrifugation. This paper provides details on the centrifuge protocol development and efficacy.     

红毛五加多糖对巨噬细胞细胞化学和超微结构的影响

本实验选用中药红毛五加多糖（ＡＧＰＳ）进行小鼠腹腔注射,对腹腔巨噬细胞细胞化学定性、定位、定量进行研究,并观察超微结构的改变,初探作用机理。实验显示了ＡＧＰＳ能使巨噬细胞数量明显增多,体积增大,细胞内糖类,酸性磷酸酶、三磷酸腺苷酶、酸性酯酶和琥珀酸脱氢酶活性显著增强。用显微分光光度计进行单细胞化学成份定量测定,实验组与对照组有显著差异,提示ＡＧＰＳ的扶正固本作用十分明显。扫描电镜显示表面微绒毛增多、增粗、增长,交错重叠或成簇、密集杂乱。透射电镜可见胞质内糖原颗粒增多、线粒体丰富、溶酶体增加,揭示了巨噬细胞活化的形态学基础。     

Effects of a gravity gradient on human cardiovascular responses

Eight subjects participated in one control and three rotation trials on a short-arm centrifuge such that the Gz levels at the feet were 0.5, 1.0, and 1.5 G. Trials consisted of 30 minutes of supine rest, 1 hour of rotation (or in the control, 30 additional minutes of rest and 30 minutes of standing), and a final 30-minute rest period. Measurements of heart rate, calf impedance, calf volume, and blood pressure support the findings that the highest G level is similar to standing and that the lower G levels fail to produce significant effects.     

Plasmalogen synthesis is regulated via alkyl-dihydroxyacetonephosphate-synthase by amyloid precursor protein processing and is affected in Alzheimer's disease

Lipids play an important role as risk or protective factors in Alzheimer's disease, which is characterized by amyloid plaques composed of aggregated amyloid-beta. Plasmalogens are major brain lipids and controversially discussed to be altered in Alzheimer's disease (AD) and whether changes in plasmalogens are cause or consequence of AD pathology. Here, we reveal a new physiological function of the amyloid precursor protein (APP) in plasmalogen metabolism. The APP intracellular domain was found in vivo and in vitro to increase the expression of the alkyl-dihydroxyacetonephosphate-synthase (AGPS), a rate limiting enzyme in plasmalogen synthesis. Alterations in APP dependent changes of AGPS expression result in reduced protein and plasmalogen levels. Under the pathological situation of AD, increased amyloid-beta level lead to increased reactive oxidative species production, reduced AGPS protein and plasmalogen level. Accordingly, phosphatidylethanol plasmalogen was decreased in the frontal cortex of AD compared to age matched controls. Our findings elucidate that plasmalogens are decreased as a consequence of AD and regulated by APP processing under physiological conditions.     

Analytical techniques for cell fractions. XVI. Preparation of protein-free supernatants with a "Z"-path rotor

A new “Z”-path centrifuge rotor is described for the separation of pellets from supernatants in up to 15 samples simultaneously. The samples and precipitating solutions are stored in separate chambers at rest, and are transferred to a sedimentation chamber by centrifugal force during rotation, where mixing occurs during rapid changes in speed, followed by sedimentation of the precipitate. The supernatant decants itself at rest into a third series of chambers, where it may be transferred centrifugally into radially disposed measuring devices, transferred with suitable pipettes, or used as a sample ring for conventional analyzers.     

红毛五加多糖对腹腔巨噬细胞作用的定量细胞化学研究

中药红毛五加(Acanthopanax giraldii Harms)属五加科植物,本文通过细胞化学定性、定位、定量研究探讨红毛五加多糖(AGPS)对腹腔巨噬细胞的作用及机理。实验证明AGPS能使巨噬细胞数量明显增多,细胞体积增大,伪足增多,吞噬能力增强,细胞内醣类、酸性磷酸酶、三磷酸腺昔酶、酸性酯酶和琥珀酸脱氢酶活性显著增强。用显微分光光度计对上述单个细胞的化学成分进行定量测定。实验组和对照组结果有显著差异。提示红毛五加的扶正固本作用十分明显,本研究为AGPS的应用和作用机理提供了一定的实验依据。     

Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices

AIMS: The aims of this study were to validate a portable continuous flow centrifuge (PCFC) as an alternative concentration step of US-EPA Method 1623 and to demonstrate it's efficacy for recovery of low numbers of protozoa from large volumes of various water matrices. METHODS AND RESULTS: Recoveries of Cryptosporidium parvum oocysts, Giardia intestinalis cysts and Encephalitozoon intestinalis spores spiked into 10-1000 l volumes of various water matrices were evaluated during in-house and collaborative trials. Spiked protozoa were either approved standards or diluted stock samples enumerated according to USEPA Method 1623. Cryptosporidium recoveries exceeded method 1623 criteria and substantially high recoveries were observed for Giardia and E. intestinalis. CONCLUSIONS: Portable continuous flow centrifuge methodology exceeded method 1623 acceptance criteria for Cryptosporidium and could be easily adopted for other protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCFC could be adopted as an alternative user-friendly concentration method for Cryptosporidium and for monitoring of large volumes of source and tap water for accidental or deliberate contamination with protozoa and potentially with other enteric pathogens. It is anticipated that PCFC would also be equal or superior to filtration for protozoa monitoring in wastewater and effluents.     

Development of medical control of man in conditions of +Gz accelerations at short-arm centrifuge

To develop a program of on-line medical monitoring of human subjects during rotation on a short-arm centrifuge (SAC), which is viewed as a promising method of providing artificial gravity, 153 runs were performed with participation of 17 subjects exposed to +Gz at 0.8, 1.2 and 1.6 G up to 40 minutes 3 times a day over a 3-d cycle. It was found that the on-line medical monitoring program for humans exposed to SAC +Gz loads should include recording of the system blood circulation and local circulation in head and leg vessels.     

Evaluation of a portable differential continuous flow centrifuge for concentration of Cryptosporidium oocysts and Giardia cysts from water

A portable device was developed and assembled from a stationary differential continuous flow centrifuge usually employed for blood cell separation, for the purpose of concentrating Cryptosporidium and Giardia from large volumes of water. Following compaction onto the wall of the disposable plastic centrifuge bowl and aspiration of residual water, the oocysts and cysts were dislodged by injection of a 20 ml solution containing 0.01% Tween-80 and 1% SDS and vigorous shaking. Following aspiration, the oocysts were pelleted, reacted with specific FITC-conjugated monoclonal antibodies, and enumerated via fluorescence microscopy. The entire procedure required about 2 h. Initially, 55% and 87% of Cryptosporidium oocysts and Giardia cysts, respectively, were recovered from 45 litres of tap water, and 27% and 57%, respectively, from river water. Adjustments in centrifuge speed and flow rates improved recovery to about 90% for Cryptosporidium oocysts and hence, this method compared favourably with the recently developed calcium carbonate flocculation method. It was superior in time requirement and volume flexibility, and showed a distinct advantage over the standard cartridge filtration method in all respects. The continuous flow centrifugation equipment is compact, mobile, flexible, and yields reproducibly high recovery rates. The ease of handling, speed of performance and minimal requirements for post-concentration equipment, reagents and labour make the system highly cost-effective. It appears to offer an improved method, well suited for use by water utilities for monitoring the burden of water-borne protozoan pathogens.     

Influence of repetitive Gz acceleration on structural and metabolic profile of m. vastus lateralis in monkeys exposed to 30 day bedrest

It was shown that changes in structural and metabolic indices of extensor muscles of the lower extremities were usually found in man after exposure to space flight or to bed rest. Similar changes were also observed in monkeys, space-flown on "Kosmos" biosatellites. Response to weightlessness and to restraint was found to be different in m. soleus and in m. vastus lateralis. Therefore, it is important to study structural and metabolic changes of m. vastus lateralis fibers under conditions of gravitational unloading in monkeys, who have motor apparatus similar to that of man, and are much more fruitful object of research. It is assumed that artificial gravity can serve as a countermeasure, aimed at diminishing effects of gravitational unloading. We have studied the effect of repeated gravity overloading, created by means of a centrifuge, on structural and metabolic indices of monkey m. vastus lateralis at the background of 30 day head down tilt bed rest (BR).     

Limitations of Stroke Volume Estimation by Non-Invasive Blood Pressure Monitoring in Hypergravity

Background

Altitude and gravity changes during aeromedical evacuations induce exacerbated cardiovascular responses in unstable patients. Non-invasive cardiac output monitoring is difficult to perform in this environment with limited access to the patient. We evaluated the feasibility and accuracy of stroke volume estimation by finger photoplethysmography (SVp) in hypergravity.

Methods

Finger arterial blood pressure (ABP) waveforms were recorded continuously in ten healthy subjects before, during and after exposure to +Gz accelerations in a human centrifuge. The protocol consisted of a 2-min and 8-min exposure up to +4 Gz. SVp was computed from ABP using Liljestrand, systolic area, and Windkessel algorithms, and compared with reference values measured by echocardiography (SVe) before and after the centrifuge runs.

Results

The ABP signal could be used in 83.3% of cases. After calibration with echocardiography, SVp changes did not differ from SVe and values were linearly correlated (p<0.001). The three algorithms gave comparable SVp. Reproducibility between SVp and SVe was the best with the systolic area algorithm (limits of agreement −20.5 and +38.3 ml).

Conclusions

Non-invasive ABP photoplethysmographic monitoring is an interesting technique to estimate relative stroke volume changes in moderate and sustained hypergravity. This method may aid physicians for aeronautic patient monitoring.     

Comparison of the starch synthesis genes between maize and rice: copies, chromosome location and expression divergence

Gene duplication and divergence are important evolutionary processes. It has been suggested that a whole genome duplication (WGD) event occurred in the Gramineae, predating its divergence, and a second WGD occurred in maize during its evolution. In this study we compared the fate of the genes involved in the core pathway of starch biosynthesis following the ancient and second WGDs in maize and rice. In total, thirty starch synthesis genes were detected in the maize genome, which covered all the starch synthesis gene families encoded by 27 genes in rice. All of these genes, except ZmGBSSIIb and ZmBEIII, are anchored within large-scale synteny blocks of rice and maize chromosomes. Previous findings and our results indicate that two of the current copies of many starch synthesis genes (including AGPL, AGPS, GBSS, SSII, SSIII, and BEII) probably arose from the ancient WGD in the Gramineae and are still present in the maize and rice genome. Furthermore, two copies of at least six genes (AGPS1, SSIIb, SSIIIb, GBSSII, BEI, and ISA3) appear to have been retained in the maize genome after its second WGD, although complete coding regions were only detected among the duplicate sets of AGPS1, SSIIb, and SSIIIb. The expression patterns of the remaining duplicate sets of starch synthesis genes (AGPL1/2, AGPS1/2, SSIIa/b, SSIIIa/b, GBSSI/II, and BEIIa/b) differ in their expression and could be classified into two groups in maize. The first group is mainly expressed in the endosperm, whereas the second is expressed in other organs and the early endosperm development. The four duplicate sets of ZmGBSSII, ZmSSIIb, ZmSSIIIb and AGPS1, which arose from the second WGD diverged in gene structure and/or expression patterns in maize. These results indicated that some duplicated starch synthesis genes were remained, whereas others diverged in gene structure and/or expression pattern in maize. For most of the duplicated genes, one of the copies has disappeared in the maize genome after the WGD and the subsequent “diploidization”. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Centrifuge Modeling of Cadmium Migration in Saturated and Unsaturated Soils

Researchers recently have come to recognize that geotechnical centrifuge can provide a powerful experimental tool for investigating the flow and transport of inorganic contaminants in soils. Inert, non-adsorptive inorganic compounds (e.g., sodium ions) rather than adsorptive heavy metals are often used as the chemical for the investigation of pollutant transport behavior in most centrifuge modeling. To overcome the shortcomings of this approach, centrifuge tests for the study of one-dimensional pollutant migration in both saturated and unsaturated soils were designed using an adsorptive pollutant (i.e., cadmium) and conducted at two centrifugal accelerations. In this study, the concentration profile of adsorptive cadmium along the depth of soil, the moisture content varying with the soil depth and the transport behavior of the cadmium in soils were investigated. The centrifuge results show that the cadmium concentration profiles are found to be similar for the centrifuge models performed at 15 g and 20 g and the one-dimensional moisture movement in the unsaturated soil can be reproducible. The validity of centrifuge modeling of adsorptive pollutants might be affected by the g-level that, in turn, determines the centrifuge testing time and affects the sorption equilibrium.     

A comparison of two centrifuge techniques for constructing vulnerability curves: insight into the ‘open‐vessel’ artifact

A vulnerability curve (VC) describes the extent of xylem cavitation resistance. Centrifuges have been used to generate VCs for decades via static‐ and flow‐centrifuge methods. Recently, the validity of the centrifuge techniques has been questioned. Researchers have hypothesized that the centrifuge techniques might yield unreliable VCs due to the open‐vessel artifact. However, other researchers reject this hypothesis. The focus of the dispute is centered on whether exponential VCs are more reliable when the static‐centrifuge method is used rather than the flow‐centrifuge method. To further test the reliability of the centrifuge technique, two centrifuges were manufactured to simulate the static‐ and flow‐centrifuge methods. VCs of three species with open vessels of known lengths were constructed using the two centrifuges. The results showed that both centrifuge techniques produced invalid VCs for Robinia because the water flow through stems under mild tension in centrifuges led to an increasing loss of water conductivity. In addition, the injection of water in the flow‐centrifuge exacerbated the loss of water conductivity. However, both centrifuge techniques yielded reliable VCs for Prunus, regardless of the presence of open vessels in the tested samples. We conclude that centrifuge techniques can be used in species with open vessels only when the centrifuge produces a VC that matches the bench‐dehydration VC.     

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min^–1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 10⁶ cells ml^–1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process.     

Effect of training for loads in the ventrodorsal direction on the blood vessels of the sympathetic trunk of the rabbit brain]

Thirty six rabbits underwent special training to get a state of being trained. In a centrifuge with a 1 m radius 18 animals got ventro-dorsal gravitation stress according to schedule N 1 (with limberung-up) and 18 animals according to schedule N 2 got gravitation stress without limbering-up. After training the animals had a 2, 3 and 4-week rest. Then a part of animals was killed and served as control and the others were subjected to a single exposure to stress of the same direction (10 units for 40 min). This stress was unendurable for untrained animals. By means of injection, clearing, measuring the vessels and histologically it was shown that the trained state was reached according to schedule N 2 and retained for 3 weeks. It was also confirmed by survival of animals and the state of the vessels of the sympathetic trunk. Training according to the 1st schedule resulted in cumulation.     

Feasibility Study of Using Centrifuge for Investigating LNAPL Migration in Unsaturated Soils

Centrifuge modeling appears potentially useful for studying geo-environmental problems such as pollutant migration in subsurface systems. In this study, the “modeling of models” technique was used to validate the feasibility of using a geotechnical centrifuge to model the transport behavior of light non-aqueous phase liquids (LNAPLs) in unsaturated soils. All the experiments were conducted to simulate a gasoline spill from a leaking underground storage tank (UST) and the subsequent subsurface migration of the gasoline. When the gravity in the centrifuge reached the desired g-level, the gasoline was released from the UST and then it migrated in the unsaturated soil corresponding to a prototype time equivalent of one year. After the centrifuge tests, soil samples were collected using sampling tubes and the concentrations of individual constituent in the LNAPL were directly measured by means of gas chromatograph analysis. Results obtained from the centrifuge tests at different g-levels show that similar migration patterns are found for LNAPL transport in unsaturated porous media. The location of the peak concentration and the behavior of lateral spreading can be adequately described. In addition, centrifuge test data show that the migration pattern of LNAPLs is related to the soil type and the physical properties of individual constituents in the LNAPLs.     

The scale-down of an industrial disc stack centrifuge

The means are described whereby a disc stack centrifuge may be scaled-down by up to 10-fold of its separation capacity. The centrifuge separation characteristics so measured are suitable for direct scale-up predictions of centrifuge performance where only small volumes of particle suspension are available for study. Such an ability to scale-down is especially important in the processing of biological particles where for example, in the early stage of process development, there is often insufficient fermentation broth for fullscale studies. Scale-down is achieved by the reduction of the number of discs available for separation purposes and by the careful positioning of these discs in the overall disc stack. A combination of dye tracer and particle separation studies are used to optimise the disc stack configuration. The resulting grade efficiency curve is an accurate reflection of the curve for the full-scale centrifuge especially in the critical design region specifying centrifuge throughput for near complete particle recovery.