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.     

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.     

The genome of bacteriophage phiKMV,a T7-like virus infecting Pseudomonas aeruginosa

The complete DNA sequence of a new lytic T7-like bacteriophage phiKMV is presented. It is the first genome sequence of a member of the Podoviridae that infects Pseudomonas aeruginosa. The linear G + C-rich (62.3%) double-stranded DNA genome of 42,519 bp has direct terminal repeats of 414 bp and contains 48 open reading frames that are all transcribed from the same strand. Despite absence of homology at the DNA level, 11 of the 48 phiKMV-encoded putative proteins show sequence similarity to known T7-type phage proteins. Eighteen open reading frame products have been assigned, including an RNA polymerase, proteins involved in DNA replication, as well as structural, phage maturation, and lysis proteins. Surprisingly, the major capsid protein completely lacks sequence homology to any known protein. Also, the strong virulence toward many clinical P. aeruginosa isolates and a short replication time make phiKMV attractive for phage therapy or a potential source for antimicrobial proteins.     

Uncertainties in alanine dosimetry in the therapeutic dose range

A method for evaluating the overall uncertainty of alanine EPR transfer dosimetry in the therapeutic dose range is described. The method uses experimental data on EPR signal reproducibility from replicate dosimeters irradiated to low doses (1–5 Gy), estimates of Type B uncertainties, and Monte Carlo simulations of heteroscedastic orthogonal linear regression. A Bruker ECS106 spectrometer and Bruker alanine dosimeters have been used for this evaluation. The results demonstrate that alanine dosimetry can be used for transfer dosimetry in that range with the overall uncertainty 1.5–4% (1σ) depending on the dose, the number of replicate dosimeters, and the duration of the calibration session (the session should not exceed one working day).     

Efficacy and safety of flavocoxid, a novel therapeutic, compared with naproxen: a randomized multicenter controlled trial in subjects with osteoarthritis of the knee

Introduction

Flavocoxid is a novel flavonoid-based “dual inhibitor” of the 5-lipoxygenase (5-LOX) enzyme and the cyclooxygenase (COX) enzymes. This study was designed to compare the effectiveness and safety of flavocoxid to naproxen in subjects with moderate to severe osteoarthritis (OA) of the knee.

Methods

In this randomized, multicenter, double-blind study, 220 subjects were assigned to receive either flavocoxid (500 mg twice daily) or naproxen (500 mg twice daily) for 12 weeks. The trial was structured to show noninferiority of flavocoxid to naproxen. Primary outcome measures included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and subscales and a timed walk.

Results

More than 90% of the subjects in both groups noted significant reduction in the signs and symptoms of knee OA. There were no statistically significant differences in efficacy between the flavocoxid and naproxen groups when the entire intent-to-treat population was analyzed. The flavocoxid group had significantly fewer upper gastrointestinal (UGI) and renal (edema) adverse events (AEs) as well as a strong trend toward fewer respiratory AEs.

Conclusion

Flavocoxid, a first-in-class flavonoid-based therapeutic that inhibits COX-1 and COX-2 as well as 5-LOX, was as effective as naproxen in managing the signs and symptoms of OA of the knee. Flavocoxid demonstrated better UGI, renal (edema), and respiratory safety profiles than naproxen.