BACKGROUND: The study aimed to evaluate the diagnostic accuracy of a new method for direct echocardiographic quantification of the myocardial infarct size, using late enhancement magnetic resonance imaging (LE-MRI) as a reference method. METHODS AND RESULTS: Echocardiography and LE-MRI were performed on average 31 days after first-time myocardial infarction in 58 patients. Echocardiography was also performed on 35 healthy controls. Direct echocardiographic quantification of the infarct size was based on automated selection and quantification of areas with hypokinesia and akinesia from colour-coded strain rate data, with manual correction based on visual wall motion analysis. The left ventricular (LV) ejection fraction, speckle-tracking-based longitudinal global strain, wall motion score index (WMSI), longitudinal systolic motion and velocity, and the ratio of early mitral inflow velocity to mitral annular early diastolic velocity were also measured by echocardiography. The area under the receiver-operating characteristic curves for the identification of the infarct size >12% by LE-MRI was 0.84, using the new method for direct echocardiographic quantification of the infarct size. The new method showed significantly a higher correlation with the infarct size by LE-MRI both at the global (r = 0.81) and segmental (r = 0.59) level compared with other indices of LV function. CONCLUSION: Direct quantification of the percentage infarct size by strain rate imaging combined with wall motion analysis yields high diagnostic accuracy and better correlation to LE-MRI compared with other echocardiographic indices of global LV function. Echocardiography performed ∼1 month after myocardial infarction showed ability to identify the patients with the infarct size >12%.

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.

A 73-year-old morbidly obese female patient (weight 125 kg, height 156 cm) was scheduled for cataract surgery of her right eye. Due to a number of severe co-morbidities general anaesthesia was contraindicated. However the patient was very anxious and requested sedation if the procedure was to be undertaken under local anaesthesia. She responded very positively to the proposal of utilising perioperative hypnosis. Hypnotic induction was achieved with the heavy eyelid technique, putting the patient into trance within 30 seconds. Continuous relaxing suggestions as described by the patient herself the day before surgery were used to maintain the trance state. She later reported that she was not aware of being in the operating room, but experienced profound relaxation during the procedure. Perioperative hypnosis proved to be a satisfactory option for sedation in this high-risk patient and should be actively considered for similar easily suggestible patients who are undergoing minor surgery.

An alternative approach to extract 3D myocardial strain based on elastic registration of the ultrasound images (3DSE) was developed by our lab. The aim of the present study was to test its clinical performance by comparing strain values obtained by 3DSE with the ones obtained with 2D speckle tracking (2DST). Standard 2D B-mode and volumetric datasets were acquired in 12 patients with coronary heart disease (CHD) and in 12 control subjects. Longitudinal (ε(LL)), circumferential (ε(CC)) and radial (ε(RR)) strain values were obtained from 2D datasets using commercially available 2DST software and from volumetric datasets using the 3DSE approach. 3DSE provided lower strain values than 2DST. With both approaches global ε(LL) and ε(CC) were significantly lower in patients with CHD than in controls. Global ε(LL) and ε(CC) correlated well between both methods (R = 0.83, R = 0.86, respectively), while segmental correlations were moderate [R = 0.63 (ε(LL)), R = 0.41 (ε(CC))]. The highest differences in ε(LL) values obtained by the two methods and the highest number of erroneous ε(LL) with 3DSE were observed in the basal LV segments. This study shows that in real-life datasets our 3DSE method provides global and regional ε(LL) and ε(CC) values that are comparable with the ones obtained from 2DST, even though they are not interchangeable with each other. As only a single acquisition is required, 3D methods may offer advantages over the current 2D techniques. However, the accuracy of the 3DSE can still be improved by solving the problems that appear with deformation estimation in the basal segments.

Cylindrospermopsis raciborskii is becoming a major concern among cyanobacteria, due to its potential ability to produce toxic metabolites. We assessed the cytotoxic potential of four C. raciborskii strains (ACT 9502, ACT 9503, ACT 9504 and ACT 9505) isolated from Lake Balaton (Hungary), by lactate dehydrogenase (LDH) leakage measurements and by detecting morphological alterations in CHO-K1 (Chinese Hamster Ovary) cells. The Australian AQS (cylindrospermopsin producer) strain of C. raciborskii and purified cylindrospermopsin (CYN) were used as positive references in both the biochemical and morphological studies. Chemical analysis for known cyanotoxins was performed on aqueous extracts of ACT and AQS strains by the HPLC-MS technique. Comparing threshold values of LDH leakage data, different toxic potentials of cyanobacterial extracts are suggested in short term (3 h) and long (24 h) exposure regimes. In the acute (3 h) experiments the aqueous extract of the ACT 9505 strain proved to be most toxic (EC(50) = 7.4 mg mL(-1)), while after 24 h the ACT 9504 extract was the most effective (EC(50) = 0.65 mg mL(-1)). The extract of the AQS strain and the purified CYN exerted most of their toxic effects after 3 h exposure (EC(50) = 0.74 mg mL(-1), and 0.9 μg mL(-1) respectively). The morphological changes of CHO-K1 cells induced by the crude extracts of the ACT strains included fragmentation of the actin filaments then relocation of the depolymerized actin to the perinuclear region, resulting cell rounding and loss of adhesion. Exposure of CHO-K1 cells to the crude extract of the AQS strain, moreover, resulted cell shrinking and formation of filopodia, i.e. distinctly different cytological alterations from that induced by the ACT extracts and the purified CYN. Chemical analysis of the cyanobacterial crude extracts confirmed the presence of cylindrospermopsin in the extract of the AQS strain (8.5 mg CYN g(-1) dry weight), and none of the presently known cyanotoxins have been analytically confirmed in the extracts of the ACT strains isolated from the Lake Balaton. Although a significant toxicity of all four ACT C. raciborskii strains is confirmed by both biochemical and morphological studies, our results also pointed out the necessity of further studies to identify the toxic, but still unknown metabolic components produced by these cyanobacterial members of the phytoplankton communities.

Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al.(2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ∼ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ∼ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.

Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of >5×10(4) s(-)(1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis.

In 2008, a successful computational design procedure was reported that yielded active enzyme catalysts for the Kemp elimination. Here, we studied these proteins together with a set of previously unpublished inactive designs to determine the sources of activity or lack thereof, and to predict which of the designed structures are most likely to be catalytic. Methods that range from quantum mechanics (QM) on truncated model systems to the treatment of the full protein with ONIOM QM/MM and AMBER molecular dynamics (MD) were explored. The most effective procedure involved molecular dynamics, and a general MD protocol was established. Substantial deviations from the ideal catalytic geometries were observed for a number of designs. Penetration of water into the catalytic site and insufficient residue-packing around the active site are the main factors that can cause enzyme designs to be inactive. Where in the past, computational evaluations of designed enzymes were too time-extensive for practical considerations, it has now become feasible to rank and refine candidates computationally prior to and in conjunction with experimentation, thus markedly increasing the efficiency of the enzyme design process.

The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chemistry.

It was previously demonstrated that secretin influenced the behavior of rats investigated by open-field test. In the present experiment, we have compared the effect of intracerebroventricular administration of 2 μg of secretin on the behavior of CFLP white and Japanese waltzing mice. These latter animals exhibit stereotypic circular movements. The effect of secretin on the horizontal (ambulation) and vertical movements (rearing and jumping) was investigated in open-field test. The ambulation time and distance were shorter, and the number of rearing and jumping were much lower in Japanese waltzing mice than in CFLP white mice during 30 min-experimental period. In white mice, 2 μg of secretin had no effect on the above-mentioned parameters; however, in Japanese waltzing mice, secretin enhanced the ambulation time and distance to the level of CFLP white mice, but did not influence the rearing and jumping. On the basis of the results, it was concluded that intracerebroventricularly administered secretin attenuated the stereotypic (circulating) movement and improved the horizontal movement indicated by the normalization of the ambulation time and distance; however, it did not influence the explorative behavior (rearing and jumping) in our special animal model.