A practical approach for the detection of DNA nanostructures in single live human cells by fluorescence microscopy.

In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells.

The primary role of glutathione against nuclear DNA damage of striatum induced by permethrin in rats.

Pyrethroids are one of the most widely used class of insecticides and their toxicity is dominated by pharmacological actions upon the CNS. This study reports as the subchronic treatment (60 days) with permethrin (PERM) (1/10 of LD(50)) induced nuclear DNA damage in rat striatum cells. Comet assay outcomes showed that PERM produced single- and double-strand breaks in striatum cells, the DNA damage was not related to oxidation at pyrimidine and purine bases. Vitamin E (280 mg/kg body weight/day) and vitamin E+coenzyme Q(10) (10 mg/kg/3 ml) supplementation could protect PERM treated rats against nuclear DNA damage. With the aim to evaluate the cause of nuclear DNA damage observed in striatum of rat treated with PERM, in vitro studies on striatum submitochondrial particles (SMPs) and on striatum cells treated with 10 muM PERM alone or plus 16 or 32 nM GSH were performed. SMPs incubated with PERM showed a decrease in superoxide anion release from the electron transport chain by inhibition of mitochondrial complex I. The effect could be related to the decrease of membrane fluidity measured in the hydrophilic-hydrophobic region of the mitochondrial membrane. This result discarded the involvement of the mitochondrial reactive oxygen species in the nuclear DNA damage. On the contrary, GSH played a crucial role on striatum since it was able to protect the cells against nuclear DNA damage induced by PERM. In conclusion our outcomes suggested that nuclear DNA damage of striatum cells was directly related to GSH depletion due to PERM insecticide.

Modification of respiratory-chain enzyme activities in brown adipose tissue mitochondria by idebenone (hydroxydecyl-ubiquinone).

Idebenone (IDE), a synthetic analog of coenzyme Q, strongly activates glycerol phosphate (GP) oxidation in brown adipose tissue mitochondria. GP oxidase, GP cytochrome c oxidoreductase and GP dehydrogenase activities were all significantly stimulated by 13 muM IDE. Substituted derivatives of IDE acetyl- and methoxyidebenone had similar activating effects. When succinate was used as substrate, no activation by IDE could be observed. The activation effect of IDE could be explained as release of the inhibition of glycerol phosphate dehydrogenase by endogenous free fatty acids. NADH oxidoreductase activity and oxidation of NADH-dependent substrates were inhibited by IDE. The extent of the inhibition and IDE concentration dependence varied when various substrates were tested, being highest for pyruvate and lowest for 2-oxoglutarate. This study thus showed that the effect of IDE on various mitochondrial enzymes is very different and thus its therapeutic use should take into account its specific effect on various mitochondrial dehydrogenases in relation to particular defects of mitochondrial respiratory chain.

Mitochondrial changes induced by natural and synthetic asbestos fibers: studies on isolated mitochondria.

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. In vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

Mitochondrial changes induced by natural and synthetic asbestos fibers: studies on isolated mitochondria.

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. in vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

Localization and mobility of coenzyme Q in lipid bilayers and membranes.

We have studied the mobility of coenzyme Q (CoQ) in lipid bilayers and mitochondrial membranes in relation to the control of electron transfer activities. A molecular dynamics computer simulation in the vacuum yielded a folded structure for CoQ10, with a length of only 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s in good agreement with those found experimentally by fluorescence quenching of pyrene derivatives. To investigate if CoQ diffusion may represent the rate-limiting step of electron transfer, we reconstituted complexes I and III and assayed the resulting NADH-cytochrome c reductase activity in presence of different CoQ10 levels and at different distances between complexes; the experimental turnovers were higher than the collision frequencies calculated using diffusion coefficients of 10(-9) cm2/s but compatible with values found by us by fluorescence quenching. Since the experimental turnovers are independent of the distance between complexes, we conclude that CoQ diffusion is not rate-limiting for electron transfer.

A high diffusion coefficient for coenzyme Q10 might be related to a folded structure.

We measured the lateral diffusion of different coenzyme Q homologues and analogues in model lipid vesicles using the fluorescence collisional quenching technique with pyrene derivatives and found diffusion coefficients in the range of 10(-6) cm2/s. Theoretical diffusion coefficients for these highly hydrophobic components were calculated according to the free volume theory. An important parameter in the free volume theory is the relative dimension between diffusant and solvent: a molecular dynamics computer simulation of the coenzymes yielded their most probable geometries and volumes and revealed surprisingly similar sizes of the short and long homologues, due to a folded structure of the isoprenoid chain in the latter, with a length for coenzyme Q10 of 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s, in good agreement with those found experimentally.

Mitochondrial complex I defects in aging.

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed. The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase. A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats. In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases.

Steady-state kinetics of reduction of coenzyme Q analogs by glycerol-3-phosphate dehydrogenase in brown adipose tissue mitochondria.

We have undertaken a study of the role of coenzyme Q (CoQ) in glycerol-3-phosphate oxidation in mitochondrial membranes from hamster brown adipose tissue, using either quinone homologs, as CoQ1 and CoQ2, or the analogs duroquinone and decylubiquinone as artificial electron acceptors. We have found that the most suitable electron acceptor for glycerol-3-phosphate:CoQ reductase activity in situ in the mitochondrial membrane is the homolog CoQ1 yielding the highest rate of enzyme activity (225 +/- 41 nmol x min(-1) x mg(-1) protein). With all acceptors tested the quinone reduction rates were completely insensitive to Complex III inhibitors, indicating that all acceptors were easily accessible to the quinone-binding site of the dehydrogenase preferentially with respect to the endogenous CoQ pool, in such a way that Complex III was kept in the oxidized state. We have also experimentally investigated the saturation kinetics of endogenous CoQ (1.35 nmol/mg protein of a mixture of 70% CoQ9 and 30% CoQ10) by stepwise pentane extraction of brown adipose tissue mitochondria and found a K(m) of the integrated activity of glycerol-3-phosphate cytochrome c reductase for endogenous CoQ of 0.22 nmol/mg protein, indicating that glycerol-3-phosphate-supported respiration is over 80% of V(max) with respect to the CoQ pool. A similar K(m) of 0.19 nmol CoQ/mg protein was found in glycerol-3-phosphate cytochrome c reductase in cockroach flight muscle mitochondria.

[Experimental isovolemic hemodilution. Study of tissue perfusion with Hb 3% in swine]. / Emodiluizione isovolemica sperimentale. Studio sulla perfusione tessutale ad Hb 3 g% nel suino.

BACKGROUND: The aim of the study is to evaluate the limits of the compensatory mechanisms and the tissue damages caused by the low oxygen content during severe normovolemic hemodilution in pigs. METHODS: The experimental procedure was performed in 10 animals after general anaesthesia was induced and iso-hypervolemic hemodilution to Hct 10% was maintained for five hours without any intensive care. Hemodynamic, biochemical and ultrastructural parameters were detected before and at the end of hemodilution in addition to analysis of oxygen delivery/uptake and mitochondrial enzymes function. RESULTS: The collected data show: the initial good compensatory mechanism was subsequently exhausted; five animals demonstrated cardiac ischemia and low CO and two of them died before the end of the experiment; no hemodynamic and hemoxymetric data predicted the cardiac ischemia; the dilution caused alterations of some detected biochemical parameters such as hemocoagulation; no evidence of morphologic and ultrastructural tissue damage or interstitial edema; decreasing in mitochondrial enzymes activity significant only for NADH-related. CONCLUSIONS: In conclusion, it seems that, in pigs at least, the compensatory mechanisms can keep a sufficient tissue oxygen supply throughout the experimental time with the exception of cardiac muscle.

The two-electron quinone reductase DT-diaphorase generates and maintains the antioxidant (reduced) form of coenzyme Q in membranes.

The experiments reported here were undertaken to test the hypothesis that the antioxidative, reduced form of hydrophobic phase coenzyme Q (CoQ) may be generated and maintained by the two-electron quinone reductase, DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] by catalyzing formation of the hydroquinone form of CoQ. This enzyme was isolated and purified from rat liver cytosol and its reduction of several CoQ homologs incorporated into large unilamellar vesicles (LUVETs) was demonstrated. The addition of NADH and DT-diaphorase to LUVETs and to multilamellar vesicles (MLVs) containing CoQ homologs, including CoQ9 and CoQ10, resulted in essentially complete reduction of the CoQ. Incorporation of either CoQ9H2 or CoQ10H2 and the lipophylic radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) into MLVs in the presence of DT-diaphorase and NADH maintained the reduced state of CoQ and inhibited lipid peroxidation. The reaction between DT-diaphorase and CoQ was also demonstrated in isolated rat liver hepatocytes in which incorporation of CoQ10 provided protection from adriamycin (adr)-induced mitochondrial membrane damage. The role of DT-diaphorase in the antioxidant activity of CoQ was demonstrated by the co-incorporation of dicoumarol (dic), a potent inhibitor of DT-diaphorase, resulting in a loss of protection by incorporated CoQ10. These results support the antioxidant function of DT-diaphorase in both artificial and natural membrane systems by acting as a two-electron CoQ reductase which forms and maintains CoQ in the reduced state.

Saturation kinetics of coenzyme Q in NADH oxidation: rate enhancement by incorporation of excess quinone.

In beef heart mitochondria it has been found that the Km for coenzyme Q10 of the NADH oxidation system is in the range of the membrane concentration of the quinone; this is contrary to succinate oxidation which is in Vmax with respect to quinone content. The same proportional difference between the two systems is maintained in their affinities for the exogenous acceptor CoQ1 in non-extracted mitochondria. The Km of succinate- coenzyme Q reductase for CoQ1 is reversibly lowered in CoQ-depleted mitochondria; while in contrast the Km for NADH-coenzyme Q reductase is reversibly increased by CoQ extraction. Incorporation of exogenous quinones by co-sonication with submitochondrial particles, as evidenced by fluorescence quenching of pyrene, enhances NADH-cytochrome c reductase activity in accordance with the lack of saturation of the former system.

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles.

The reduction kinetics of coenzyme Q (CoQ, ubiquinone) by NADH:ubiquinone oxidoreductase (complex I, EC 1.6.99.3) was investigated in bovine heart mitochondrial membranes using water-soluble homologs and analogs of the endogenous ubiquinone acceptor CoQ10 [the lower homologs from CoQ0 to CoQ3, the 6-pentyl (PB) and 6-decyl (DB) analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ1 and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ1 and PB as acceptors. The kinetic pattern follows a ping-pong mechanism; the Km for CoQ1 is in the range of 20 microM but is reversibly increased to 60 microM by extraction of the endogenous CoQ10. The increased Km in CoQ10-depleted membranes indicates that endogenous ubiquinone not only does not exert significant product inhibition but rather is required for the appropriate structure of the acceptor site. The much lower Vmax with CoQ2 but not with DB as acceptor, associated with an almost identical Km, suggests that the sites for endogenous ubiquinone bind 6-isoprenyl- and 6-alkylubiquinones with similar affinity, but the mode of electron transfer is less efficient with CoQ2. The Kmin (kcat/Km) for CoQ1 is 4 orders of magnitude lower than the bimolecular collisional constant calculated from fluorescence quenching of membrane probes; moreover, the activation energy calculated from Arrhenius plots of kmin is much higher than that of the collisional quenching constants. These observations strongly suggest that the interaction of the exogenous quinones with the enzyme is not diffusion-controlled. Contrary to other systems, in bovine submitochondrial particles, CoQ1 usually appears to be able to support a rate approaching that of endogenous CoQ10, as shown by application of the "pool equation" [Kröger, A., & Klingenberg, M. (1973) Eur. J. Biochem. 39, 313-323] relating the rate of ubiquinone reduction to the rate of ubiquinol oxidation and the overall rate through the ubiquinone pool.

Major changes in complex I activity in mitochondria from aged rats may not be detected by direct assay of NADH:coenzyme Q reductase.

We have investigated the respiratory activities and the concentrations of respiratory chain components of mitochondria isolated from the livers and hearts of two groups of rats aged 6 and 24 months respectively. In comparison with the adult controls (6 months), in aged rats there was a decline in total aerobic NADH oxidation in both tissues; only minor (non-significant) changes, however, were found in NADH:coenzyme Q reductase and cytochrome oxidase activities, and there was no change in ubiquinol-cytochrome c reductase activity. The coenzyme Q levels were slightly decreased in mitochondria from both organs of aged rats. The lowered NADH oxidase activity is not due to the slight decrease observed in the coenzyme Q levels, but is the result of decreased Complex I activity. Since the assay of NADH:coenzyme Q reductase requires quinone analogues, none of which can evoke its maximal turnover [Estornell, Fato, Pallotti and Lenaz (1993) FEBS Lett. 332, 127-131], its activity has been calculated indirectly by taking advantage of the relationship that exists between NADH oxidation and ubiquinol oxidation through the coenzyme Q pool. The results, expressed in this way, show a drastic loss of activity of Complex I in both the heart and the liver of aged animals in comparison with adult controls.

Underevaluation of complex I activity by the direct assay of NADH-coenzyme Q reductase in rat liver mitochondria.

We have shown that the rate of NADH-coenzyme Q reductase in rat liver mitochondria, assayed using the decyl-ubiquinone analog DB, is underevaluated, probably as a result of its low water solubility. In view of drawbacks encountered using other more soluble acceptors in this system, we demonstrate that the most reliable assay of the physiological rate of CoQ reduction by Complex I is the indirect calculation from the total rate of NADH oxidation and the rate of ubiquinol oxidation, using the pool equation of Kröger and Klingenberg [(1973) Eur. J. Biochem. 34, 358-368].

Mitochondrial activities of rat heart during ageing.

Some analytical and functional parameters of rat heart mitochondrial have been investigated at six different periods of ageing from 2 to 26 months. The fatty acid composition of the mitochondrial membranes reveals a percentage increase of polyunsaturated fatty acids (20:4 n-6, 22:6 n-3) up to 12 months, followed by a decrease; however, fluorescence polarization of the membrane probe diphenylhexatriene is not changed, revealing that membrane fluidity is not significantly affected. No major change in ubiquinone-9 and in cytochrome content is apparent, indicating that the relative ratio of the respiratory chain components is unmodified. Nevertheless, significant changes in enzyme specific activities are detected: NADH cytochrome c reductase and cytochrome oxidase activities increase up to 12 months, then decrease at 18-26 months; ubiquinol cytochrome c reductase exhibits a peak at 18 months, followed by a decrease. All these activities follow a similar trend during the whole life span of the rat, even though the 'maximum' is different. No significant changes have been found in ATP synthase. Succinate-cytochrome c reductase steadily increases over the whole life span. The results, showing activity decreases in the respiratory enzymes having subunits encoded by mitochondrial DNA, are compatible with the 'mitochondrial' theory of ageing.

Protective effect of exogenous coenzyme Q against damage by adriamycin in perfused rat liver.

We have investigated the effect of rat liver perfusion with adriamycin on mitochondrial activities. Although the perfusion treatment per se induces some decline of respiratory activities, adriamycin strongly potentiates this effect; moreover the coenzyme Q9 content of the mitochondrial membrane is significantly lowered by the antibiotic. Coaddition of coenzyme Q10 in the perfusate significantly protects the mitochondria, not only from loss of respiratory activities but also of the endogenous CoQ9 content. Exogenous CoQ10 fails to enhance respiratory activities in control rats, not treated with adriamycin, even though CoQ concentration has been proven not to be kinetically saturating in the respiratory chain under physiological conditions. Thus, the beneficial effect of CoQ10 in the perfusate does not appear to be the result of its role in the respiratory chain but is a consequence of its antioxidant action.

An updating of the biochemical function of coenzyme Q in mitochondria.

The apparent Km for coenzyme Q10 in NADH oxidation by coenzyme Q (CoQ)-extracted beef heart mitochondria is close to their CoQ content, whereas both succinate and glycerol-3-phosphate oxidation (the latter measured in hamster brown adipose tissue mitochondria) are almost saturated at physiological CoQ concentration. Attempts to enhance NADH oxidation rate by excess CoQ incorporation in vitro were only partially successful: the reason is in the limited amount of CoQ10 that can be incorporated in monomeric form, as shown by lack of fluorescence quenching of membrane fluorescent probes; at difference with CoQ10, CoQ5 quenches probe fluorescence and likewise enhances NADH oxidation rate above normal. Attempts to enhance the CoQ content in perfused rat liver and in isolated hepatocytes failed to show uptake in the purified mitochondrial fraction. Nevertheless CoQ cellular uptake is able to protect mitochondrial activities. Incubation of hepatocytes with adriamycin induces loss of respiration and mitochondrial potential measured in whole cells by flow cytometry using rhodamine 123 as a probe: concomitant incubation with CoQ10 completely protects both respiration and potential. An experimental study of aging in the rat has shown some decrease of mitochondrial CoQ content in heart, and less in liver and skeletal muscle. In spite of the little change observed, it is reasoned that CoQ administration may be beneficial in the elderly, owing to the increased demand for antioxidants.

Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase.

The assay of Complex I activity requires the use of artificial acceptors, such as short-chain coenzyme Q homologs and analogs, because the physiological quinones, such as CoQ10, are too insoluble in water to be added as substrates to the assay media. The medical interest raised in the last years on the pathological changes of Complex I activity has focussed on the requirement of easy reliable assays for its analysis. We have undertaken a systematic examination of the assay conditions of Complex I in mitochondrial membranes, using a series of quinones as electron acceptors, particularly the coenzyme Q homologs CoQ0, CoQ1 and CoQ2, and the analogs duroquinone and decylubiquinone. Our findings have pointed out that the most suitable electron acceptor for the NADH:CoQ reductase assay is the homolog CoQ1. The analog DB, commercially available, although yielding a high activity, nevertheless causes some problems for the standardization of the assay conditions.