Assessing physical activity in people with mental illness: 23-country reliability and validity of the simple physical activity questionnaire (SIMPAQ).

BACKGROUND: Physical inactivity is a key contributor to the global burden of disease and disproportionately impacts the wellbeing of people experiencing mental illness. Increases in physical activity are associated with improvements in symptoms of mental illness and reduction in cardiometabolic risk. Reliable and valid clinical tools that assess physical activity would improve evaluation of intervention studies that aim to increase physical activity and reduce sedentary behaviour in people living with mental illness. METHODS: The five-item Simple Physical Activity Questionnaire (SIMPAQ) was developed by a multidisciplinary, international working group as a clinical tool to assess physical activity and sedentary behaviour in people living with mental illness. Patients with a DSM or ICD mental illness diagnoses were recruited and completed the SIMPAQ on two occasions, one week apart. Participants wore an Actigraph accelerometer and completed brief cognitive and clinical assessments. RESULTS: Evidence of SIMPAQ validity was assessed against accelerometer-derived measures of physical activity. Data were obtained from 1010 participants. The SIMPAQ had good test-retest reliability. Correlations for moderate-vigorous physical activity was comparable to studies conducted in general population samples. Evidence of validity for the sedentary behaviour item was poor. An alternative method to calculate sedentary behaviour had stronger evidence of validity. This alternative method is recommended for use in future studies employing the SIMPAQ. CONCLUSIONS: The SIMPAQ is a brief measure of physical activity and sedentary behaviour that can be reliably and validly administered by health professionals.

A complex four-point method for the evaluation of ohmic and faradaic losses within a redox flow battery single-cell.

We propose a complex 4-point method for characterization of flow batteries. The distribution of ohmic and faradaic losses within a single-cell is evaluated from electrochemical impedance spectra and load curves of positive and negative half-cells measured with platinum wire pseudo-reference electrodes positioned in respective electrode compartment. The developed method can be used e.g., for the component screening and in-situ durability studies on single-cell scale. The method was validated on a vanadium redox flow battery single-cell; however, it can be analogically employed for various chemistries of flow battery. •Complex 4-point method for characterization of flow battery single-cell was developed.•Method is based on electrochemical impedance spectra and load curve measurements.•Direct evaluation of ohmic and faradaic losses distribution within battery single-cell by the method.

Dehydration Preparation of Mouse Sperm for Vitrification and Rapid Laser Warming.

BACKGROUND: Mice are fundamental models of study due to their ease of breeding, manipulation, and the well-studied genome. There has been extensive research focused on the cryopreservation of mouse germaplasm, as a way to help maintain the different transgenic mouse breeds. The first protocols for mouse sperm were developed in the 90's using slow cooling and a mixture of raffinose and glycerol. Since then, the rate of success reported remains highly variable. OBJECTIVE: The Aim of this work is to study factors that are key for developing vitrification protocols for ultra-rapid laser warming of mouse sperm. RESULTS: Our results show that due to the exquisite sensitivity of sperm cells to osmotic excursions, our target levels of dehydration (~85% water content) cannot be achieved without causing a significant decrease in sperm motility and membrane fusion. CONCLUSION: It seems likely that mouse sperm vitrification is going to be difficult to develop due to the exquisite sensitivity of mouse sperm cells to handling and dehydration.

Factors influencing quality of anticoagulation control and warfarin dosage in patients after aortic valve replacement within the 3 months of follow up.

Warfarin dosage estimation using the pharmacogenetic algorithms has been shown to improve the quality of anticoagulation control in patients with atrial fibrillation. We sought to assess the genetic, demographic and clinical factors that determine the quality of anticoagulation in patients following aortic valve replacement (AVR). We studied 200 consecutive patients (130 men) aged 63 ± 12.3 years, undergoing AVR, in whom warfarin dose was established using a pharmacogenetic algorithm. The quality of anticoagulation within the first 3 months since surgery was expressed as the time of international normalized ratio (INR) in the therapeutic range (TTR). The median TTR in the entire cohort was 59.6% (interquartile range, 38.7 - 82.7). Ninety-nine (49.5%) patients with TTR ≥ 60% did not differ from those with poor anticoagulation control (TTR < 60%) with regard to demographic and cardiovascular risk factors. Coronary artery disease (n = 84, 42%) and previous stroke (n = 5, 2.5%) predicted higher TTR, while possession of CYP2C9*2 variant allele (n = 49, 25%) was associated with lower TTR (P = 0.01). In turn, VKORC1 c.-1639A, CYP2C9*2 and *3 variants were independently associated with actual warfarin dose (P < 0.0001). In AVR patients better anticoagulation control is observed in patients with coronary artery disease and history of stroke, which might result in part from previous lifestyle modification and therapy. Possession of CYP2C9*2 and/or CYP2C9*3 allele variants is associated with lower TTR values and warfarin dose variations in AVR patients, the latter affected also by VKORC1 c.-1693G>A polymorphism.

Influence of the surface properties on bactericidal and fungicidal activity of magnetron sputtered Ti-Ag and Nb-Ag thin films.

In this study the comparative investigations of structural, surface and bactericidal properties of Ti-Ag and Nb-Ag thin films have been carried out. Ti-Ag and Nb-Ag coatings were deposited on silicon and fused silica substrates by magnetron co-sputtering method using innovative multi-target apparatus. The physicochemical properties of prepared thin films were examined with the aid of X-ray diffraction, grazing incidence X-ray diffraction, scanning electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy methods. Moreover, the wettability of the surface was determined. It was found that both, Ti-Ag and Nb-Ag thin films were nanocrystalline. In the case of Ag-Ti film presence of AgTi3 and Ag phases was identified, while in the structure of Nb-Ag only silver occurred in a crystal form. In both cases the average size of crystallites was ca. 11 nm. Moreover, according to scanning electron microscopy and atomic force microscopy investigations the surface of Nb-Ag thin films was covered with Ag-agglomerates, while Ti-Ag surface was smooth and devoid of silver particles. Studies of biological activity of deposited coatings in contact with Bacillus subtilis, Pseudomonas aeruginosa, Enterococcus hirae, Klebisiella pneumoniae, Escherichia coli, Staphylococcus aureus and Candida albicans were performed. It was found that prepared coatings were bactericidal and fungicidal even in a short term-contact, i.e. after 2 h.

Determination of structural, mechanical and corrosion properties of Nb2O5 and (NbyCu 1-y)Ox thin films deposited on Ti6Al4V alloy substrates for dental implant applications.

In this paper comparative studies on the structural, mechanical and corrosion properties of Nb2O5/Ti and (NbyCu1-y)Ox/Ti alloy systems have been investigated. Pure layers of niobia and niobia with a copper addition were deposited on a Ti6Al4V titanium alloy surface using the magnetron sputtering method. The physicochemical properties of the prepared thin films were examined with the aid of XRD, XPS SEM and AFM measurements. The mechanical properties (i.e., nanohardness, Young's modulus and abrasion resistance) were performed using nanoindentation and a steel wool test. The corrosion properties of the coatings were determined by analysis of the voltammetric curves. The deposited coatings were crack free, exhibited good adherence to the substrate, no discontinuity of the thin film was observed and the surface morphology was homogeneous. The hardness of pure niobium pentoxide was ca. 8.64GPa. The obtained results showed that the addition of copper into pure niobia resulted in the preparation of a layer with a lower hardness of ca. 7.79 GPa (for niobia with 17 at.% Cu) and 7.75 GPa (for niobia with 25 at.% Cu). The corrosion properties of the tested thin films deposited on the surface of titanium alloy depended on the composition of the thin layer. The addition of copper (i.e. a noble metal) to Nb2O5 film increased the corrosion resistance followed by a significant decrease in the value of corrosion currents and, in case of the highest Cu content, the shift of corrosion potential towards the noble direction. The best corrosion properties were obtained from a sample of Ti6Al4V coated with (Nb0.75Cu0.25)Ox thin film. It seems that the tested materials could be used in the future as protection coatings for Ti alloys in biomedical applications such as implants.

Opposing role of Notch1 and Notch2 in a Kras(G12D)-driven murine non-small cell lung cancer model.

Lung cancer is the leading cause of cancer-related deaths worldwide. Recently, we have shown that Notch1 inhibition resulted in substantial cell death of non-small cell lung cancer (NSCLC) cells in vitro. New compounds targeting Notch signal transduction have been developed and are now being tested in clinical trials. However, the tumorigenic role of individual Notch receptors in vivo remains largely unclear. Using a Kras(G12D)-driven endogenous NSCLC mouse model, we analyzed the effect of conditional Notch1 and Notch2 receptor deletion on NSCLC tumorigenesis. Notch1 deficiency led to a reduced early tumor formation and lower activity of MAPK compared with the controls. Unexpectedly, Notch2 deletion resulted in a dramatically increased carcinogenesis and increased MAPK activity. These mice died significantly earlier due to rapidly growing tumor burden. We found that Notch1 regulates Ras/MAPK pathway via HES1-induced repression of the DUSP1 promoter encoding a phosphatase specifically suppressing pERK1/2. Interestingly, Notch1 but not Notch2 ablation leads to decreased HES1 and DUSP1 expression. However, Notch2-depleted tumors showed an appreciable increase in ß-catenin expression, a known activator of HES1 and important lung cancer oncogene. Characteristically for ß-catenin upregulation, we found that the majority of Notch2-deficient tumors revealed an undifferentiated phenotype as determined by their morphology, E-Cadherin and TTF1 expression levels. In addition, these carcinomas showed aggressive growth patterns with bronchus invasion and obstruction. Together, we show that Notch2 mediates differentiation and has tumor suppressor functions during lung carcinogenesis, whereas Notch1 promotes tumor initiation and progression. These data are further supported by immunohistochemical analysis of human NSCLC samples showing loss or downregulation of Notch2 compared with normal lung tissue. In conclusion, this is the first study characterizing the in vivo functions of Notch1 and Notch2 in Kras(G12D)-driven NSCLC tumorigenesis. These data highlight the clinical importance of a thorough understanding of Notch signaling especially with regard to Notch-targeted therapies.

Nɛ-homocysteinyl-lysine isopeptide is associated with progression of peripheral artery disease in patients treated with folic acid.

OBJECTIVE: Folic acid (FA) administration can reduce plasma total homocysteine (tHcy); however, it fails to decrease cardiovascular events and progression of peripheral artery disease (PAD). Nɛ-homocysteinyl-lysine isopeptide (Nɛ-Hcy-Lys) is formed during catabolism of homocysteinylated proteins. We sought to investigate factors that determine the presence of Nɛ-Hcy-Lys in PAD patients with hyperhomocysteinemia receiving FA. PATIENTS AND METHODS: We studied 131 consecutive PAD patients with tHcy > 15 µmol l(-1) taking FA 0.4 mg d(-1) for 12 months. Serum Nɛ-Hcy-Lys was determined by high-performance liquid chromatography (HPLC). We also measured interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1), asymmetric dimethylarginine (ADMA) and 8-iso-prostaglandin F(2α) (8-iso-PGF(2α)). RESULTS: FA administration resulted in a 70.5% decrease in tHcy (p < 0.0001). However, serum Nɛ-Hcy-Lys was detectable in 28 (21.4%) patients on FA who were more frequently current smokers and survivors of ischaemic stroke (p < 0.001). They had higher tHcy by 46.0%, PAI-1 by 51.7%, 8-iso-PGF(2α) by 59.1% and ADMA by 26.4% (all, p < 0.0001). The presence of Nɛ-Hcy-Lys was associated with lower ankle-brachial index (ABI) values (p < 0.001) and higher prevalence of cardiovascular events (p < 0.001) following therapy. CONCLUSION: The presence of Nɛ-Hcy-Lys in one-fifth of hyperhomocysteinemic individuals with PAD despite FA treatment is associated with progression of PAD and with increased ADMA formation, oxidative stress and hypofibrinolysis.

Laboratory constraints on chameleon dark energy and power-law fields.

We report results from a search for chameleon particles created via photon-chameleon oscillations within a magnetic field. This experiment is sensitive to a wide class of unexplored chameleon power-law and dark energy models. These results exclude 5 orders of magnitude in the coupling of chameleons to photons covering a range of 4 orders of magnitude in chameleon effective mass and, for individual models, exclude between 4 and 12 orders of magnitude in chameleon couplings to matter.

Search for chameleon particles using a photon-regeneration technique.

We report the first results from the GammeV search for chameleon particles, which may be created via photon-photon interactions within a strong magnetic field. Chameleons are hypothesized scalar fields that could explain the dark energy problem. We implement a novel technique to create and trap the reflective particles within a jar and to detect them later via their afterglow as they slowly convert back into photons. These measurements provide the first experimental constraints on the couplings of chameleons to photons.

Search for axionlike particles using a variable-baseline photon-regeneration technique.

We report the first results of the GammeV experiment, a search for milli-eV mass particles with axionlike couplings to two photons. The search is performed using a "light shining through a wall" technique where incident photons oscillate into new weakly interacting particles that are able to pass through the wall and subsequently regenerate back into detectable photons. The oscillation baseline of the apparatus is variable, thus allowing probes of different values of particle mass. We find no excess of events above background and are able to constrain the two-photon couplings of possible new scalar (pseudoscalar) particles to be less than 3.1x10;(-7) GeV-1 (3.5x10;(-7) GeV-1) in the limit of massless particles.

Coral larvae conservation: physiology and reproduction.

Coral species throughout the world's oceans are facing severe environmental pressures. We are interested in conserving coral larvae by means of cryopreservation, but little is known about their cellular physiology or cryobiology. These experiments examined cryoprotectant toxicity, dry weight, water and cryoprotectant permeability using cold and radiolabeled glycerol, spontaneous ice nucleation temperatures, chilling sensitivity, and settlement of coral larvae. Our two test species of coral larvae, Pocillopora damicornis (lace coral), and Fungia scutaria (mushroom coral) demonstrated a wide tolerance to cryoprotectants. Computer-aided morphometry determined that F. scutaria larvae were smaller than P. damicornis larvae. The average dry weight for P. damicornis was 24.5%, while that for F. scutaria was 17%, yielding osmotically inactive volumes (V(b)) of 0.22 and 0.15, respectively. The larvae from both species demonstrated radiolabeled glycerol uptake over time, suggesting they were permeable to the glycerol. Parameter fitting of the F. scutaria larvae data yielded a water permeability 2 microm/min/atm and a cryoprotectant permeability = 2.3 x 10(-4) cm/min while modeling indicated that glycerol reached 90% of final concentration in the larvae within 25 min. The spontaneous ice nucleation temperature for F. scutaria larvae in filtered seawater was -37.8+/-1.4 degrees C. However, when F. scutaria larvae were chilled from room temperature to -11 degrees C at various rates, they exhibited 100% mortality. When instantly cooled from room temperature to test temperatures, they showed damage below 10 degrees C. These data suggest that they are sensitive to both the rate of chilling and the absolute temperature, and indicate that vitrification may be the only means to successfully cryopreserve these organisms. Without prior cryopreservation, both species of coral settled under laboratory conditions.

High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option.

Although fish embryos have been used in a number of slow-freezing cryopreservation experiments, they have never been successfully cryopreserved. In part this is because little is known about whether ice forms within the embryo during the slow-freezing dehydration process. Therefore, we examined the temperature of intraembryonic ice formation (T(IIF)) and the temperature of extraembryonic ice formation (T(EIF)), using a cryomicroscope. We used both unmodified zebrafish embryos and those with water channels (aquaporin-3 or AQP3) inserted into their membranes to increase permeability to water and cryoprotectants, examined at 100% epiboly to the 6-somite stage. In these experiments we examined: (1) the spontaneous freezing of (external) solutions; (2) the spontaneous freezing of solutions containing embryos; (3) the effect of preloading the embryos with cryoprotectants on T(IIF); (4) whether preloading the embryos with cryoprotectant helps in survival after nucleating events in the solution; and (5) the damaging effects of extracellular nucleation events versus solution toxicity on the embryos. The solutes alone (embryo medium--EM, sucrose culture medium, 1 M propylene glycol in EM, and 1 M propylene glycol in a sucrose culture medium) froze at -14.9 +/- 1.1, -17.0 +/- 0.3, -17.8 +/- 1.0, and -17.7 +/- 1.4, respectively. There was no difference amongst these means (P > 0.05), thus adding cryoprotectant did not significantly lower the nucleation point. Adding embryos (preloaded with cryoprotectant or not) did not change the basic freezing characteristics of these solutes. In all these experiments, (T(EIF)) equaled (T(IIF)), and there was no difference in the freezing point of the solutions with or without the embryos (P > 0.05). Additionally, there was no difference in the freezing characteristics of embryos with and without aquaporins (P > 0.05). The formation of intraembryonic ice was lethal to the zebrafish embryos in all cases. But this lethal outcome was not related to solution injury effects, because 88-98% of embryos survived when exposed to a higher solute concentration with no ice present. Taken together, these data suggest that slow-freezing is not a suitable option for zebrafish embryos. The mechanism of this high temperature nucleation event in zebrafish embryos is still unknown.

Expression and characterization of protein geranylgeranyltransferase type I from the pathogenic yeast Candida albicans and identification of yeast selective enzyme inhibitors.

Protein geranylgeranyltransferase type I (GGTase I) is a heterodimeric zinc metalloenzyme catalyzing protein geranylgeranylation at cysteine residues present in C-terminal signature sequences referred to as CaaX (X=Leu) motifs. We have studied GGTase I as a potential antifungal target and recently reported its purification and cloning from the yeast Candida albicans (Ca GGTase I), an important human pathogen. Here, we report the high yield bacterial expression of Ca GGTase I by coexpression of maltose binding protein fusion proteins of both the alpha (Ram2p) and beta (Cdc43p) subunits. The cleaved and purified recombinant Ca GGTase I was demonstrated to be functional and structurally intact as judged by the presence of one equivalent of a tightly bound zinc atom and the near stoichiometric formation, isolation and catalytic turnover of a geranylgeranyl pyrophosphate-GGTase I complex. Kinetic analysis was performed with a native substrate protein, Candida Cdc42p, which exhibited significant pH dependent substrate inhibition, a feature not observed with other Ca GGTase I substrates. Prenyl acceptor substrate specificity was studied with a series of peptides in which both the CaaX motif, and the sequence preceding it, were varied. The prenyl acceptor K(M)s were found to vary nearly 100-fold, with biotinyl-TRERKKKKKCVIL, modeled after a presumably geranylgeranylated Candida protein, Crl1p (Rho4p), being the optimal substrate. A screen for inhibitors of Ca GGTase I identified compounds showing selectivity for the Candida versus human GGTase I. The most potent and selective compound, L-689230, had an IC(50) of 20 nM and >12,500-fold selectivity for Ca GGTase I. The lack of significant anti-Candida activity for any of these inhibitors is consistent with the recent finding that GGTase I is not required for C. albicans viability [R. Kelly et al., J. Bacteriol. 182 (2000) 704-713].

The enhancement of the ability of mouse sperm to survive freezing and thawing by the use of high concentrations of glycerol and the presence of an Escherichia coli membrane preparation (Oxyrase) to lower the oxygen concentration.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have hypothesized several underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (<0.3 M) than the approximately 1 M concentrations that are optimal for most mammalian cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have been able to obtain high motilities in 0.8 M glycerol provided that the exposure time is held to approximately 5 min to minimize toxicity and provided that the glycerol is added and removed stepwise to minimize osmotic shock. Since free radical damage in mouse sperm is proportional to the oxygen concentrations, we have determined the consequences of reducing the oxygen to <3% of atmospheric by maintaining the sperm in contact with an Escherichia coli membrane preparation, Oxyrase, from the moment of collection throughout the assessment of motility. Prior studies have shown that the procedure significantly reduces damage from centrifugation and osmotic shock. In the experiments reported here we obtained approximately 50% motility relative to untreated controls when suspensions containing 3.8% Oxyrase were exposed approximately 5 min to a solution of 0.8 M glycerol and 0.17 M (10%) raffinose in a supplemented PBS and then frozen at approximately 25 degrees C/min to -75 degrees C. In the absence of Oxyrase, the normalized motility dropped to 31%. The protection by Oxyrase was in part a consequence of minimizing centrifugation damage, but in part it reflected a reduction in freeze-thaw damage. Preliminary experiments indicate that the number of motile sperm after cryopreservation in Oxyrase is higher when the sperm are collected without swim-up than when they are collected by swim-up. This is in part due to the fact that more cells are collected in the absence of swim-up and in part due to a greater protective effect of Oxyrase on those cells. The minimum temperature in these initial experiments was limited to -75 degrees C to avoid the potential contribution of other injurious factors between -75 and -196 degrees C.

Geranylgeranyltransferase I of Candida albicans: null mutants or enzyme inhibitors produce unexpected phenotypes.

Geranylgeranyltransferase I (GGTase I) catalyzes the transfer of a prenyl group from geranylgeranyl diphosphate to the carboxy-terminal cysteine of proteins with a motif referred to as a CaaX box (C, cysteine; a, usually aliphatic amino acid; X, usually L). The alpha and beta subunits of GGTase I from Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and each is essential for viability. We are evaluating GGTase I as a potential target for antimycotic therapy of the related yeast, Candida albicans, which is the major human pathogen for disseminated fungal infections. Recently we cloned CaCDC43, the C. albicans homolog of S. cerevisiae CDC43. To study its role in C. albicans, both alleles were sequentially disrupted in strain CAI4. Null Cacdc43 mutants were viable despite the lack of detectable GGTase I activity but were morphologically abnormal. The subcellular distribution of two GGTase I substrates, Rho1p and Cdc42p, was shifted from the membranous fraction to the cytosolic fraction in the cdc43 mutants, and levels of these two proteins were elevated compared to those in the parent strain. Two compounds that are potent GGTase I inhibitors in vitro but that have poor antifungal activity, J-109,390 and L-269,289, caused similar changes in the distribution and quantity of the substrate. The lethality of an S. cerevisiae cdc43 mutant can be suppressed by simultaneous overexpression of RHO1 and CDC42 on high-copy-number plasmids (Y. Ohya et al., Mol. Biol. Cell 4:1017, 1991; C. A. Trueblood, Y. Ohya, and J. Rine, Mol. Cell. Biol. 13:4260, 1993). Prenylation presumably occurs by farnesyltransferase (FTase). We hypothesize that Cdc42p and Rho1p of C. albicans can be prenylated by FTase when GGTase I is absent or limiting and that elevation of these two substrates enables them to compete with FTase substrates for prenylation and thus allows sustained growth.

Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose.

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.

Factors affecting yield and survival of cells when suspensions are subjected to centrifugation. Influence of centrifugal acceleration, time of centrifugation, and length of the suspension column in quasi-homogeneous centrifugal fields.

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.

Influence of centrifugation regimes on motility, yield, and cell associations of mouse spermatozoa.

Mouse sperm are exceptionally sensitive to mechanical forces associated with pipetting and mixing. This characteristic raised the question of the sensitivity of mouse sperm to centrifugation, a step necessary in the removal of cryoprotectants and a common component in the general manipulation of sperm suspensions for experimental purpose. Epididymal spermatozoa from ICR mice were isolated and manipulated to minimize pipetting and mixing damage. The centrifugal accelerations studied were 200, 400, 600, and 800 x g (measured with a stroboscope) for 5, 10, or 15 minutes of centrifugation time. The number of cells and the number of motile cells were counted. The percent motility and longevity, total yield, and motile yield were calculated. Centrifugation at 200 and 400 x g for short times (5 minutes) caused only a small loss in either immediate or 2.5-hour motility, but centrifugation at 600 and 800 x g for 15 minutes produced up to a fivefold loss. Low speed/short time centrifugation pelleted only about half of the cells; the others were lost when the supernatant was removed. The maximum number of motile sperm (motile yield) was obtained at intermediate centrifugal forces (approximately 400 x g for 10-12 minutes), and it is the total number of motile sperm (and not the percent motility) that is important in the use of cryopreserved sperm to regenerate cryopreserved mutant lines. Relative centrifugal force and centrifugation time exhibit reciprocity (e.g., 200 x g for 10 minutes produces similar results to 400 x g for 5 minutes). The spermatozoa must be centrifuged under carefully defined conditions to minimize the damage and to maximize the recovery of viable cells.