Protein crystallization: from HTS to kilogram-scale

The first experiments on protein crystallization started randomly during the 19th century. This technique has been widely used for the determination of the tertiary structure of proteins since the 1950s, when an understanding of the physics of protein crystallization began to emerge. In the 1980s and 1990s, research focused on the study of protein crystal growth processes in microgravity environments, which were created in space shuttle experiments. High-throughput screening (HTS) systems were developed that later found broader laboratory applications. The combination of HTS with an engineering approach opens new opportunities for the protein crystallization process to become a robust, scalable, reproducible and economically viable industrial unit operation.     

Difficult laryngoscopy: incidence and predictors in patients undergoing coronary artery bypass surgery versus general surgery patients

OBJECTIVE: Cardiac surgery patients might have a higher incidence of difficult laryngoscopy than the general population because of older age, dental problems, and obesity. The authors estimated the incidence and predictors of difficult laryngoscopy in coronary artery bypass surgery patients. DESIGN: Prospective, controlled study. SETTING: University setting. PARTICIPANTS: Patients undergoing coronary artery bypass or general surgery. INTERVENTIONS: Two hundred consecutive patients undergoing coronary artery bypass graft and 444 general surgery patients, all aged >40 years, were compared for the incidence and predictors of difficult laryngoscopy, defined as a grade III or IV view. MEASUREMENTS AND MAIN RESULTS: Predictors of difficult laryngoscopy were considered mouth opening <4 cm, limited cervical mobility, thyromental distance <6 cm, protruding or partially missing upper teeth, and Mallampati classes 3 and 4. More cases of difficult laryngoscopy were recorded in cardiac patients (10% v 5.2%, p <0.023). The cardiac patients were older, mostly men, and belonged to ASA III-IV risk classes. Mallampati classes 3 and 4 were more frequent in the control group. With univariate analysis, difficult laryngoscopy correlated with 7 variables: older age, ASA-IV risk class, protruding or partially missing upper teeth, limited mouth opening, limited neck movement, thyromental distance <6 cm, and diabetes mellitus. Multivariate analysis adjusted for propensity score identified older age (odds ratio = 1.05/yr, 95% confidence interval = 1.005-1.09, p < 0.03) and limited neck movement (odds ratio = 9.5, 95% confidence interval = 2.2-41, p < 0.003), but not cardiac surgery per se, as independent predictors of difficult laryngoscopy. CONCLUSIONS: Difficult laryngoscopy was more frequent in cardiac surgery patients (10% v 5.2%). Older age and limited neck movement, but not cardiac surgery per se, were independent predictors of difficult laryngoscopy.     

Modulation of electroencephalographic responses to transcranial magnetic stimulation: evidence for changes in cortical excitability related to movement

Transcranial magnetic stimulation (TMS) and multichannel electroencephalography (EEG) were used for the investigation of cortical excitability preceding voluntary movement in human subjects. The study showed the practical value of the combined TMS-EEG approach in differentiating between cortical and spinal-cord mechanisms, which is difficult with conventional electromyographic measures alone. TMS induced a pronounced negativity (N100) lasting for 150-200 ms, with the amplitude maximum in the stimulated hemisphere. When TMS was applied just before the onset of the visually triggered movement, N100 was markedly attenuated, although motor evoked potentials (MEPs) became larger. We suggest that the N100 component represents an inhibitory response following TMS. This interpretation is in agreement with intracellular recordings in animals, paired-pulse TMS studies and experiments showing increased premovement excitability on the basis of MEPs. N100 was not affected only by the subsequent movement, but also by the switching from rest to the motor-task condition, which caused a slight attenuation of the N100 component; no changes, however, were found in the amplitude of MEPs, suggesting that modified excitability did not affect the output of the corticospinal pyramidal cells. By contrast to MEPs, N100 was modulated also by the presentation of the visual stimulus alone, i.e. when no movement was required. This attenuation suggests that even in a rest condition visual stimuli have an access to the sensorimotor regions of the cortex, most probably through ascending arousal brain systems.     

Brain injury and neurofunctional deficit in neonatal mice with hypoxic-ischemic encephalopathy

Birth asphyxia accounts for the majority of developmental motor and cognitive deficits. Studies were undertaken to develop a reproducible murine model of perinatal hypoxic-ischemic encephalopathy (HIE) which would permit both anatomic and neurofunctional quantification of injury. Short-term neurofunctional outcomes consisted of three developmental reflexes (righting, cliff aversion and geotaxis) assessed in 7-day-old mouse pups 24 h after unilateral carotid artery ligation followed by inhalation of 8% oxygen. Cerebral infarct volume was dependent on duration of hypoxia, being approximately 2.5-fold greater with longer (60 min) versus shorter (30 min) hypoxia exposure (P=0.001). All three sensorimotor neonatal reflexes assessed at 24 h after HIE injury correlated significantly with long-term neurofunction evaluated using a water-maze test of navigational learning and memory assessed 8 weeks later in the same animals. Cerebral atrophy, a delayed consequence of HIE in this model, also correlated strongly with water-maze performance (r=0.86, P=0.002). These data demonstrate for the first time that murine neonatal sensorimotor reflex performance can be rigorously quantified in the acute phase of perinatal HIE and has strong predictive value not only for anatomic extent of cerebral injury, but also for long-term neurofunctional outcome.     

"Binge" cocaine differentially alters preproenkephalin mRNA levels in guinea pig brain

Male Hartley guinea pigs were administered i.p. injections of cocaine or saline for 2 or 7 days in a "binge" paradigm. RNA was isolated from dissected brain regions and levels of preproenkephalin mRNA and total RNA were quantified by RNase protection assays. Following 2 days of "binge" cocaine administration, no significant alterations in preproenkephalin mRNA levels were detected in six brain regions. Following 7 days of cocaine administration, however, lower levels of preproenkephalin mRNA were observed in the nucleus accumbens and hypothalamus of cocaine-treated animals and higher levels in the frontal cortex and amygdala. These findings differed from previous studies in the rat, so an additional experiment was performed with animals treated at the 7 day time point. For increased statistical power, data from the two experiments were combined and examined by two-way ANOVAs; in this combined analysis, increases in preproenkephalin mRNA were observed in frontal cortex, amygdala, and hippocampus, decreases were found in the nucleus accumbens and hypothalamus, with no change in thalamus, caudate putamen, or cerebellum. These observed differences between guinea pigs and rats make this species an interesting model for neurobiological studies of cocaine-induced alterations in neuropeptide gene expression in the mammalian brain.     

Elevation of guinea pig brain preprodynorphin mRNA expression and hypothalamic-pituitary-adrenal axis activity by “binge” pattern cocaine administration

The endogenous opioid system and the hypothalamic-pituitary-adrenal (HPA) axis have been implicated in many of the neurobiological effects of cocaine. Previous studies in our laboratory showed that “binge” pattern cocaine administration increases preprodynorphin (ppDyn) mRNA levels in the caudate putamen and circulating levels of corticosterone in the rat. The present study extended these findings to guinea pigs, a species known to have a κ opioid receptor profile similar to that of humans. Male guinea pigs were treated with: (a) “binge” pattern cocaine for 7 days (subchronic) (3 × 15 mg/kg/day, hourly, intraperitoneal); (b) “binge” pattern saline for 5 days followed by “binge” pattern cocaine for 2 days (subacute); or (c) “binge” pattern saline for 7 days. Thirty minutes after the final injection, levels of ppDyn mRNA were quantitated in the nucleus accumbens, caudate putamen, frontal cortex, amygdala, hippocampus, and hypothalamus using a solution hybridization RNase protection assay. Regional distribution of ppDyn mRNA levels in the guinea pig brain was similar to that found in rat, with highest levels in the nucleus accumbens and caudate putamen. In the caudate putamen, ppDyn mRNA was significantly increased following either 2 days (38% increase) or 7 days (32% increase) of “binge” pattern cocaine administration as compared to saline-treated controls. No significant changes in ppDyn mRNA levels were found in any other brain region. Both subacute and subchronic “binge” cocaine administration significantly elevated plasma levels of adrenocorticotropin hormone (ACTH) and cortisol. However, the ACTH and cortisol increases were significantly blunted following 7 days of “binge” cocaine administration as compared to 2 days of drug treatment, reflecting the development of HPA tolerance or adaptation to repeated cocaine administration. Thus, the ppDyn mRNA and HPA responses to cocaine in guinea pigs are similar to those observed in rats.     

Basal metabolic rate in the Yakut (Sakha) of Siberia.

Human indigenous circumpolar populations have elevated basal metabolic rates (BMRs) relative to predicted values; this metabolic elevation has been postulated to be a physiological adaptation to chronic and severe cold stress. The present study examines BMR in the Yakut, an indigenous high-latitude population from the Sakha Republic of Russia to determine (1) whether the Yakut show evidence of an elevated BMR, (2) if the Yakut display evidence of age-related changes in BMR, and (3) whether lifestyle differences influence BMR. BMR was measured during the late summer in 75 women and 50 men (ages 18-56 years) from the Siberian village of Berdygestiakh. Measured BMR (+/- SEM) of the entire sample was significantly elevated (+6.5%) compared to predictions based on body mass (6,623.7 +/- 94.9 vs. 6,218.2 +/- 84.7 kJ/day; P < 0.001). Additionally, measured BMR for the entire sample was significantly higher than predictions based on fat-free mass (+20.8%) and surface area (+8.9%). Males and females both showed significant elevations relative to all three standards. The elevated BMR of the Yakut does not appear to be attributable to extreme levels of protein, since the Yakut consume a mixed diet with a substantial proportion of carbohydrates. No significant age-related changes in BMR were found when controlled for body composition. No significant relationship was found between lifestyle variables and BMR, suggesting the possibility of a genetic or developmental mechanism. This study provides additional evidence of metabolic elevation in indigenous circumpolar groups and has important implications for estimating the nutritional requirements of these populations.