The effects of refreezing on the viscoelastic and tensile properties of ligaments

Biomechanical testing protocols for ligaments can be extensive and span two or more days. During this time, a specimen may have to undergo more than one cycle of freezing and thawing. Thus, the objective of this study was to evaluate the effects of refreezing on the viscoelastic and tensile properties of ligaments. The femur-medial collateral ligament-tibia complexes (FMTC) from six pairs of rabbit knees were used for this study. Following sacrifice, one leg in each pair was assigned to the fresh group and the FMTC was immediately dissected and prepared for testing. The contralateral knees were fresh-frozen at -20 degrees C for 3 weeks, thawed, dissected and then refrozen for one additional week before being tested as the refrozen group. The cross-sectional area and shape of the medial collateral ligament (MCL) was measured using a laser micrometer system. Stress relaxation and cyclic stress-relaxation tests in uniaxial tension were performed followed by a load to failure test. When the viscoelastic behavior of the MCL was described by the quasi-linear viscoelastic (QLV) theory, no statistically significant differences could be detected for the five constants (A, B, C, tau1, and tau2) between the fresh and refrozen groups (p > or = 0.07) based on our sample size. In addition, the structural properties of the FMTCs and the mechanical properties of the MCLs were also found to be similar between the two groups (p > or = 0.68). These results suggest that careful refreezing of the specimens had little or no effect on the biomechanical properties measured.     

Temperature-dependent viscoelastic properties of the human supraspinatus tendon

Temperature effects on the viscoelastic properties of the human supraspinatus tendon were investigated using static stress-relaxation experiments and the quasi-linear viscoelastic (QLV) theory. Twelve supraspinatus tendons were randomly assigned to one of two test groups for tensile testing using the following sequence of temperatures: (1) 37, 27, and 17 °C (Group I, n=6), or (2) 42, 32, and 22 °C (Group II, n=6). QLV parameter C was found to increase at elevated temperatures, suggesting greater viscous mechanical behavior at higher temperatures. Elastic parameters A and B showed no significant difference among the six temperatures studied, implying that the viscoelastic stress response of the supraspinatus tendon is not sensitive to temperature over shorter testing durations. Using regression analysis, an exponential relationship between parameter C and test temperature was implemented into QLV theory to model temperature-dependent viscoelastic behavior. This modified approach facilitates the theoretical determination of the viscoelastic behavior of tendons at arbitrary temperatures.     

The effects of tibial-femoral angle on the failure mechanics of the canine anterior cruciate ligament

A series of canine femur-ACL-tibia complexes were subjected to tensile tests with axial tibial orientation and 0 degree, 45 degrees or 90 degrees femoral orientation with respect to load direction. A deflection rate of 51.0 cm min-1 was used in all tests. Marked differences occurred in ultimate loads, deflection and energy absorbed as a consequence of differences in femoral-tibial orientation. The mode of structural failure, as determined by post-test examination, also varied markedly as a function of femoral-tibial orientation. It is concluded that differences both in measured mechanical properties and observed failure details are a consequence of varying the loading pattern of the fiber bundles across the finite breadth of the ligament.     

Ternary complex formation of human immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an intermediate of membrane fusion

Human immunodeficiency virus (HIV) Env-induced fusion is highly temperature dependent. When effector and target cells were coincubated at 37 degrees C, there was a kinetic delay before fusion commenced. When effector and target cells were coincubated for varied times at 23 degrees C, a temperature that does not permit fusion, a temperature-arrested stage was created. Raising temperature to 37 degrees C from the 23 degrees C intermediate eliminated the kinetic delay. Inhibitors (T22, AMD3100, and Sch-C) that block fusion by binding chemokine receptors were added after creating the intermediate so as to assess the extent of engagement between gp120 and chemokine receptors at that stage. For both CXCR4 and CCR5 as coreceptors, increasingly long times of coincubation at 23 degrees C reduced the efficacy of the coreceptor-binding inhibitors in blocking fusion. This implies that an increasing number of ternary Env/CD4/coreceptor complexes form over time at 23 degrees C. It also shows that ternary complex formation has a lower temperature threshold than the downstream steps that include Env folding into a six-helix bundle; this provides an experimental means to separate coreceptor binding by gp120 from the subsequent refolding of gp41 into a six-helix bundle structure. As the time of cell coincubation at 23 degrees C was prolonged, more cells quickly fused upon the raising of the temperature to 37 degrees C, and the increase quantitatively correlated with the greater percentage of fusion that was resistant to drugs. Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form.     

Heat-induced changes in the mechanics of a collagenous tissue: isothermal, isotonic shrinkage.

We present data from isothermal, isotonic-shrinkage tests wherein bovine chordae tendineae were subjected to well-defined constant temperatures (from 65 to 90 degrees C), durations of heating (from 180 to 3600 s), and isotonic uniaxial stresses during heating (from 100 to 650 kPa). Tissue response during heating and "recovery" at 37 degrees C following heating was evaluated in terms of the axial shrinkage, a gross indicator of underlying heat-induced denaturation. There were three key findings. First, scaling the heating time via temperature and load-dependent characteristic times for the denaturation process collapsed all shrinkage data to a single curve, and thereby revealed a time-temperature-load equivalency. Second, the characteristic times exhibited an Arrhenius-type behavior with temperature wherein the slopes were nearly independent of applied load--this suggested that applied loads during heating affect the activation entropy, not energy. Third, all specimens exhibited a time-dependent, partial recovery when returned to 37 degrees C following heating, but the degree of recovery decreased with increases in the load imposed during heating. These new findings on heat-induced changes in tissue behavior will aid in the design of improved clinical heating protocols and provide guidance for the requisite constitutive formulations.     

Effects of temperature on the transport of nucleosides in guinea pig erythrocytes

The initial rate of [14C]uridine transport by guinea pig erythrocytes was investigated at different temperatures. At 37, 22, and 10 degrees C the concentration dependence of uridine zero-trans influx and equilibrium exchange influx was resolved into two components; (a) a saturable component which followed simple Michaelis-Menten kinetics and which was inhibited by nitrobenzylthioinosine, and (b) a linear component of low magnitude and insensitive to nitrobenzylthioinosine inhibition. The maximum velocity, Vmax, of zero-trans uridine influx for the saturable transport system was 70-fold higher at 37 than 10 degrees C (1.24, 0.20, and 0.018 mmol/L of cells per hour at 37, 22, and 10 degrees C, respectively). Similarly, the apparent affinity, Km, for zero-trans influx decreased as the temperature was lowered (0.27, 0.066, and 0.038 mM at 37, 22, and 10 degrees C, respectively). In contrast, uridine equilibrium exchange influx was less temperature dependent (Vmax, 2.80, 0.89, and 0.14 mmol/L of cells per hour; apparent Km 0.61, 0.36, and 0.24 mM at 37, 22, and 10 degrees C, respectively). These results demonstrate that the mobility of the empty carrier is impaired to a greater extent than the mobility of the loaded carrier temperature decreased. However, the kinetic constants for zero-trans uridine influx and efflux at 37 degrees C were similar, indicating that the nucleoside transporter exhibited directional symmetry at 37 degrees C. Arrhenius plots of the maximum velocity for equilibrium exchange and zero-trans uridine influx were discontinuous above 25 degrees C, but between 20 and 5 degrees C the plots were linear (Ea = 22 and 30 kcal/mol for equilibrium exchange and zero-trans influx, respectively.     

The effect of ambient temperature on beta-adrenergic responsiveness in rats.

The responses of tail skin and colonic temperatures of female rats to ambient temperatures of 20, 22, 24, 26, 28, and 30 degrees C were measured. Within this range, colonic temperature was stable while tail skin temperature increased linearly with increasing ambient temperature. Administration of the beta-adrenergic agonist, d,l-isoproterenol, at 10.0, 25.0, and 62.5 micrograms/kg, sc, at each ambient temperature was accompanied by increases in tail skin and colonic temperatures that were dependent on both the dose of isoproterenol administered and the ambient temperature. The integrated responses of tail skin temperature following administration of the three doses of isoproterenol were maximal at an ambient temperature of 26 degrees C while the integrated responses of colonic temperature were maximal at 30 degrees C. The results suggest that tests of beta-adrenergic responsiveness using this technique should be performed at an ambient temperature of 26 degrees C for maximal sensitivity.     

Effect of morphological features and surface area of hydroxyapatite on the fatigue behavior of hydroxyapatite-polyethylene composites

The effect of surface area and morphological features of filler particles on the fatigue behavior of hydroxyapatite-filled high-density polyethylene composites was studied. Composites containing 40 vol % filler were injection-molded into tensile bars, gamma-irradiated, and subjected to sinusoidal tensile fatigue at a frequency of 2 Hz. To simulate the physiological environment, the tests were conducted at 37 degrees C in saline. Results showed that properties such as secant modulus, cyclic energy dissipation, dynamic creep strain, hysteresis loops, and even fracture surfaces differ depending on the morphology and surface area of the fillers used.     

Comparative study of the precipitation of low density lipoproteins by aortic proteodermatan sulphate and heparin

The aortic proteoglycans and heparin were shown to form insoluble complexes with human low density lipoproteins (LDL). The effect of temperature, polyethylene glycol and ionic strength on the formation of complexes between porcine aortic proteodermatan sulphate (PDS) and LDL has been studied by laser nephelometry and comparisons made with heparin LDL complexes. Turbidity was a nonlinear function of the quantity of LDL precipitated by PDS. The turbidity of aggregates was constant at temperatures between 2 degrees C and 30 degrees C but increased with temperature above 30 degrees C up to 50 degrees C. The formation of insoluble complexes decreased rapidly with increasing NaCl concentration. Polyethylene glycol enhanced the turbidity at 20 degrees C but not at 37 degrees C. It also increased the resistance of complexes to dissociation by increasing ionic strength. The turbidity of heparin--LDL complexes was linearly correlated with the quantity of precipitated LDL. The heparin-LDL aggregates were less sensitive to modification of temperature and ionic strength than the PDS-LDL aggregates. These results suggest that ionic interactions are weaker in PDS-LDL complexes than in the heparin-LDL complexes. Non-coulombic interactions and/or temperature dependent conformational changes may be involved in the stabilization of supramolecular PDS-LDL aggregates. No such interactions or changes appear to be involved in complex formation between heparin and LDL.     

Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures?

Our primary objective was to determine if rates of fluid-phase endocytosis (FPE) were conserved in hepatocytes from organisms acclimated and adapted to different temperatures. To this aim, the fluorescent dye Lucifer yellow was employed to measure FPE at different assay temperatures (AT) in hepatocytes from 5 degrees C- and 20 degrees C-acclimated trout, Oncorhynchus mykiss (at 5 and 20 degrees C AT), 22 degrees C- and 35 degrees C-acclimated tilapia, Oreochromis nilotica (at 22 and 35 degrees C AT), and the Sprague-Dawley rat (at 10, 20, and 37 degrees C AT). FPE was also studied in rats fed a long-chain polyunsaturated fatty acid (PUFA)-enriched diet (at 10 degrees C AT). Despite being temperature dependent, endocytic rates (values in pl. cell(-1). h(-1)) in both species of fish were compensated after a period of acclimation. For example, in 20 degrees C-acclimated trout, the rate of endocytosis declined from 1.84 to 1.07 when the AT was reduced from 20 to 5 degrees C; however, after a period of acclimation at 5 degrees C, the rate (at 5 degrees C AT) was largely restored (1.80) and almost perfectly compensated (95%). In tilapia, endocytic rates were also temperature compensated, although only partially (36%). Relatively similar rates obtained at 5 degrees C in 5 degrees C-acclimated trout (1.8), at 20 degrees C in 20 degrees C-acclimated trout (1.84), and at 22 degrees C in 22 degrees C-acclimated tilapia (2.2) suggest that endocytic rates are somewhat conserved in these two species of fish. In contrast, the rate in rat measured at 37 degrees C (16.83) was severalfold greater than in fish at their respective body temperatures. A role for lipids in determining rates of endocytosis was supported by data obtained at 10 degrees C in hepatocytes isolated from rats fed a long-chain PUFA-enriched diet: endocytic rates were higher (5.35 pl. cell(-1). h(-1)) than those of rats fed a standard chow diet (2.33 pl. cell(-1). h(-1)). The conservation of endocytic rates in fish may be related to their ability to conserve other membrane characteristics (i.e., order or phase behavior) by restructuring their membrane lipid composition or by modulating the activities of proteins that regulate endocytosis and membrane traffic, whereas the lack of conservation between fish and rat may be due to differences in metabolic rate.     

Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents

beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase.     

Strongyloides ratti: thermokinetic behavior of third-stage larvae on a temperature gradient

We examined the thermokinetic behaviors of infective third-stage larvae (L3) of the rodent parasitic nematode Strongyloides ratti on temperature gradients using an in vitro agarose tracking assay method. Observed behaviors included both negative and positive thermokineses, the direction of movement depending both on the gradient temperature at which larvae were initially placed and on prior experience of culture temperature. Larvae isolated from rat feces cultured at 25 degrees C and placed on a gradient at temperatures between 22 degrees and 29 degrees C tended to move toward higher temperatures. At higher placement temperatures, most larvae moved little and showed no directional response, whereas at lower placement temperatures, many migrated toward cooler temperatures. At placement temperatures of 20 degrees C or below, few or no larvae moved toward the zone of higher temperature. Larvae isolated from rat feces cultured at 20 degrees C tended to migrate to a high temperature area regardless of placed temperature. Those cultured at 30 degrees C did not respond to the temperature gradient. L3 cultured at 30 degrees C were significantly less infective to rats than those cultured at 25 degrees or 20 degrees C. Additional experiments were designed to demonstrate thermokinetic behaviors during the period after reaching the L3 stage. Larvae incubated in double distilled water (DDW) for 24 h at 37 degrees C lost their ability to respond to lower temperatures, while in those incubated in DDW at 15 degrees and 25 degrees C, responses were still apparent. The thermokinetic behavior of S. ratti L3 is affected by surrounding environmental temperatures and this may have an important role in host finding.     

The effect of incubation temperature on the maintenance of rat palatal mucosa in organ culture

The effect of incubation temperature on the behavior of neonatal rat palatal mucosa maintained in a chemically defined medium in organ culture for periods up to 7 days was investigated. Explant survival was optimal at 37 degrees C with increasing mortality at temperatures of 34 degrees C and 30 degrees C. There was a transient increase in the epithelial mitotic activity at all temperatures, but at all time intervals mitotic activity was greatest at 37 degrees C. While the mitotic activity at 37 degrees C after 5 hr in vitro was comparable with previously described in vivo values, it was subsequently increased, only returning to values approximating those at the start of the experiment at 6 days. At 30 degrees and 34 degrees C the epithelial mitotic activity increased more slowly than at 37 degrees C; then it followed a similar pattern with time and after 5 days in vitro had fallen to values approximating initial values. At the cut edges of the explants, the rate of epithelial migration and subsequent keratinization increased with increasing temperature. It is suggested that survival of neonatal rat palatal mucosa is optimal in this organ culture system when maintained at 37 degrees C.     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Sensitivity of Encephalitozoon cuniculi to various temperatures, disinfectants and drugs.

Spores of Encephalitozoon cuniculi were exposed to various temperature or to disinfectants, and their infectivity was then tested on monolayer cultures of canine kidney cells. The maximum survival time for spores suspended in medium 199 was 1 day at -20 degrees C, 98 days at 4 degrees C, 6 days at 22 degrees C, and 2 days at 37 degrees C. Only 2.5% survived 30 min at 56 degrees C. Boiling for 5 min or autoclaving at 120 degrees C for 10 min killed all spores. Dry spores survived less than a week at 4 degrees C but at least 4 weeks at 22 degrees C. Exposure for 30 min to recommended working concentrations of 9 of the 11 disinfectants tested killed all spores. The growth-inhibition effect of 7 antibiotics and chemotherapeutics was studied on canine kidney cell culture inoculated with E. cuniculi. None could completely inhibit growth. The most effective was chloroquine phosphate which, at a concentration of 12.5 mg per 1000 ml culture medium and during a test period of 8 weeks, reduced the harvest of E. cuniculi to 31% of that from inoculated, untreated cultures.     

Water content alters viscoelastic behaviour of the normal adolescent rabbit medial collateral ligament.

Testing environment is an important factor in the outcome of mechanical tests on connective tissue. The purpose of this investigation was to determine the effect of ligament water content on ligament mechanical behaviour by altering the test environment. Water content of medial collateral ligament (MCLs) from 19 three-month-old New Zealand White rabbits was varied in subsets of ligaments pairs by means of immersion in 2, 10 or 25% sucrose or 0.9% phosphate-buffered saline (PBS) solutions for 1 h. One knee joint was cycled 50 times in the designated solution (experimental), while the contralateral knee (uncycled control) was simultaneously soaked in the same tank. Following cycling, the water contents of both test and control ligaments were determined. Water contents of 22 normal MCLs were determined immediately post-sacrifice and served as 'normal water content' controls. Normalized peak cyclic load changes were used as a measure of the viscoelastic behaviour of each MCL. Results demonstrated that only ligaments soaked (but not cycled) in a 10% sucrose solution had water contents (60.5 +/- 2.5%) which were statistically similar to the 22 fresh normal MCLs (63.9 +/- 6.0%). Ligaments soaked in PBS (74.0 +/- 1.3%) or 2% sucrose (69.2 +/- 2.3%) had significantly higher water contents compared to fresh normal MCLs. Ligaments with higher water contents (e.g. soaked in PBS or 2% sucrose) demonstrated greater cyclic load relaxation compared to ligaments with lower contents (e.g. soaked in 25 or 10% sucrose). Different fluid test environments can significantly alter ligament water content and, in turn, significantly affect ligament viscoelastic behaviour.     

The quality standardization of a national vaccine against yellow fever

One of the commercial lots of yellow fever vaccine has been attested as the National Branch Standard (NBS) of yellow fever vaccine. This NBS has been studied in all tests required by the regulations for standard vaccines. The NBS has been found to meet the necessary requirements in all its characteristics. The study of the thermostability of the NBS at temperatures of 4-10 degrees C, 20-22 degrees C, 37 degrees C during storage for 24 hours to 1 year has revealed the rapid loss of the infectious capacity of the virus at the above temperatures and its high stability during storage at -20 degrees C. Thus, the NBS has been found to retain the required level of immunizing potency for 3 months at a temperature of 4-10 degrees C, for 1 month at 20-22 degrees C and for 2 weeks (the term of observation) at 37 degrees C. The heat resistance of the NBS of yellow fever vaccine corresponds to the WHO requirements. The newly developed NBS has been used as the standard preparation for controlling 27 lots of commercial yellow fever vaccine.     

Temperature-dependent induction of thermotolerance by ethanol

Ethanol (1 M) cytotoxicity in asynchronous Chinese hamster ovary cells was strongly temperature dependent, yielding families of cell survival curves between 34 and 39 degrees C that were similar to those obtained at hyperthermic temperatures in medium without ethanol. Below 36 degrees C, survival curves were biphasic, indicating the development of thermotolerance during ethanol exposures. At room temperature (22 degrees C) ethanol was completely nontoxic with incubation periods up to 6 h. A comparison of survival curves with and without ethanol showed that the major effect of ethanol was an effective temperature shift of circa 6.5 degrees C, i.e., the cell survival curve at 37 degrees C in 1 M ethanol was equivalent to that at 43.6 degrees C in medium without ethanol. In addition to the effective temperature shift, ethanol also resulted in sensitization to "heat" with a temperature dependence that was similar to the stepdown heating effect. When thermotolerance was induced with acute ethanol exposures (25 min, 37 degrees C or 60 min, 35.5 degrees C), the kinetics and the magnitude of tolerance were similar to those after isotoxic conditioning treatments with heat alone (10 min, 45 degrees C). In contrast, equimolar ethanol at 22 degrees C did not induce thermotolerance. These data provide a rationale for conflicting results in the literature regarding thermotolerance induction by ethanol. Both heat sensitization and the induction of thermotolerance are interpreted as the effect of ethanol on the solution properties of intracellular water. These solvent alterations reduce the temperature necessary to elicit cytotoxicity and the development of thermotolerance.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)