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Objective: In this study, the authors aimed to evaluate the effectiveness of the vena cava distensibility index and pulse pressure variation as dynamic parameters for estimating intravascular volume in critically ill children. Methods: Patients aged 1 month to 18 years, who were hospitalized in the present study's pediatric intensive care unit, were included in the study. The patients were divided into two groups according to central venous pressure: hypovolemic (< 8 mmHg) and non-hypovolemic (central venous pressure ≥ 8 mmHg) groups. In both groups, vena cava distensibility index was measured using bedside ultrasound and pulse pressure variation. Measurements were recorded and evaluated under arterial monitoring. Results: In total, 19 (47.5%) of the 40 subjects included in the study were assigned to the central venous pressure ≥ 8 mmHg group, and 21 (52.5%) to the central venous pressure < 8 mmHg group. A moderate positive correlation was found between pulse pressure variation and vena cava distensibility index (r = 0.475, p < 0.01), while there were strong negative correlations of central venous pressure with pulse pressure variation and vena cava distensibility index (r = –0.628, p < 0.001 and r = –0.760, p < 0.001, respectively). In terms of predicting hypovolemia, the predictive power for vena cava distensibility index was > 16% (sensitivity, 90.5%; specificity, 94.7%) and that for pulse pressure variation was > 14% (sensitivity, 71.4%; specificity, 89.5%). Conclusion: Vena cava distensibility index has higher sensitivity and specificity than pulse pressure variation for estimating intravascular volume, along with the advantage of non-invasive bedside application.

The reduction of failure rates of small-sized zirconia devices depends on the understanding of their micromechanical properties. This paper reports the micromechanical behaviors of porous and dense zirconia materials using in-situ micropillar compression with a flat diamond indenter in a scanning electron microscope (SEM). Porous and dense zirconia micropillars were made using focused ion beam (FIB) milling technique in the SEM. They were then subject to in-situ SEM compression to identify their Young's moduli, yield stresses, plastic deformation, compressive and fracture strengths, damage accumulations, and failure mechanisms. We found that while both porous and dense zirconia microstructures exhibited plastic behaviors, the former had much lower Young's moduli, strengths (yield, compression and fracture), resilience and toughness but higher ductility, resulting in significant buckling than the latter. In plastic regions, alternative strain softening and hardening may have caused stress variations in porous zirconia while dislocation movement contributed to strain hardening in dense zirconia. Although both zirconia materials had quasi-brittle failures, there were different damage mechanisms. The quasi-brittle failure for porous state was due to mushrooming buckling damage driven by breaking of weak interconnected pore networks, resulting in severe compaction and pulverization, microcracks and material piling. The quasi-brittle failure for dense state was identified as plastic crushing damage, involving microcrack initiation and propagation, cleavage and intergranular fractures, and delamination. The mechanical properties of porous and dense zirconia micropillars investigated contributed to the knowledge on deformation and damage mechanisms of zirconia materials at the small scale., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Efficient diamond machining of zirconia requires a comprehensive understanding of repetitive diamond indentation mechanics. This paper reports on in-situ cyclic nanoindentations of pre-sintered and sintered zirconia materials performed inside a scanning electron microscope (SEM). In-situ SEM imaging of cyclic indentation processes and high-magnification SEM mapping of indentation imprints were conducted. The elastic and plastic behaviors of pre-sintered and sintered zirconia materials were investigated as a function of the cyclic nanoindentation number using the Sakai and Sakai-Nowak models. For pre-sintered zirconia, cyclic nanoindentation induced quasi-plastic deformation, causing localized agglomeration of zirconia crystals with microcracks and large cracking along the indentation edge. Severely compressed, fragmented, and pulverized zirconia crystals and smeared surfaces were also observed. For sintered zirconia, shear bands dominated quasi-plastic deformation with the formation of edge pile-ups and localized microfractures occurred at indentation apex and diagonals. All elastic and plastic behaviors for pre-sintered and sintered zirconia materials revealed significantly microstructure-dependent. Pre-sintered zirconia yielded significantly lower contact hardness, Young's moduli, resistance to plasticity, elastic deformation components, and resistance to machining-induced cracking, and higher elastic and plastic displacements than sintered state. Meanwhile, all the behaviors for the two materials were independent from the cyclic nanoindentation number. A model was proposed for cyclic nanoindentation mechanics, revealing their cyclic indentation-induced microstructural changes in the two zirconia materials. This study advances the fundamental understanding of nanoindentation mechanics of zirconia materials., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Development of optimal shaping processes for pre-sintered and sintered zirconia materials requires a fundamental understanding of damage and deformation mechanisms at small-scale contacts with diamond tools. This paper reports on responses of zirconia materials with distinct microstructures to nanoindentation associated with diamond machining using a Berkovich diamond indenter. In-situ nanoindentation was performed in a scanning electron microscope (SEM) and in-process filmed to record small contact events. Indentation morphology was SEM-mapped at high-magnifications. Although both pre-sintered porous and sintered dense zirconia materials mechanically revealed the quasi-plastic behavior in indentation, there were distinct responses of the two materials to quasi-plasticity at the microstructural level. For pre-sintered porous zirconia, the quasi-plasticity was attributed to shear faults resulting from breaking pore networks as microstructurally discrete interfaces, to lead to compression, fragmentation, pulverization and microcracking of zirconia crystals in indentation imprints. In contrast, sintered dense zirconia had shear band-induced quasi-plastic deformation, accompanied with localized tensile microfracture. A material index associated with the mechanical properties ranked the lower quasi-plasticity for pre-sintered porous zirconia than its sintered dense state, predicting more machining-induced damage in the former than the latter. Significantly higher indentation imprint volumes induced in indented pre-sintered porous zirconia than sintered dense state previses higher machining efficiency for the former than the latter. The microstructure-dependent indentation mechanisms provide the fundamental knowledge into micromechanics of abrasive machining of zirconia materials and may lead to a new microstructural design for zirconia materials to achieve a balanced machining efficiency and damage control., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation., (Copyright © 2011. Published by Elsevier Ltd.)

Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.

Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock. The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin). The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7. Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH. These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it. Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains. In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin., (Copyright 2002 Elsevier Science Ltd.)

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of alpha-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd approximately 175-250 microM at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic methods., (Copyright 1999 Academic Press.)

Human glucose 6-phosphate dehydrogenase (G6PD) has a particularly large number of variants resulting from point mutations; some 60 mutations have been sequenced to date. Many variants, some polymorphic, are associated with enzyme deficiency. Certain variants have severe clinical manifestations; for such variants, the mutant enzyme almost always displays a reduced thermal stability. A homology model of human G6PD has been built, based on the three-dimensional structure of the enzyme from Leuconostoc mesenteroides. The model has suggested structural reasons for the diminished enzyme stability and hence for deficiency. It has shown that a cluster of mutations in exon 10, resulting in severe clinical symptoms, occurs at or near the dimer interface of the enzyme, that the eight-residue deletion in the variant Nara is at a surface loop, and that the two mutations in the A- variant are close together in the three-dimensional structure.

Alpha-toxin of Clostridium perfringens, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20 degrees C. The crystals diffract to a minimum Bragg spacing of 2.7 A, belong to the space group R32 (with a = b = 153.3 A, c = 95.4 A, alpha = beta = 90 degrees and gamma = 120 degrees) and contain a single polypeptide chain in the crystallographic unit.

NAD+ dependent D(-)-mandelate dehydrogenase from the yeast Rhodotorula graminis strain KGX 39 has been crystallized in three different forms using the hanging drop vapour diffusion method at 15 to 20 degrees C. Type I crystals belong to space group P222(1), P22(1)2(1) or P2(1)2(1)2(1) with a = 100.3 A, b = 117.4 A, c = 80.4 A and are likely to contain a dimer in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P22(1)2(1) or P2(1)2(1)2(1) with a = 187.8 A, b = 122.9 A, c = 72.1 A and contain probably two dimers in the crystallographic asymmetric unit. They diffract to dmin = 1.8 A. Type III crystals belong to space group P2(1)2(1)2(1) with a = 109.6, b = 52.0 A, c = 145.7 A, and are likely to contain a dimer in the crystallographic asymmetric unit. They diffract at least to dmin = 2.5 A.

Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.