Removal methods of rigid stylets to minimise adverse force and tracheal tube movement: a mathematical and in-vitro analysis in manikins.

This study investigated displacement of the tracheal tube caused by different methods of intubating stylet removal, using in-vitro experiments and mathematical analysis. In the first in-vitro experiment, we measured the distance travelled by the tube tip during stylet extraction. Then, we investigated the ideal technique for stylet extraction using mathematical analysis, which would cause minimal tube displacement. Then, using a training manikin, we measured the force applied to the vocal cords and stylet extraction force during tracheal intubation. When the stylet was extracted along a straight path towards the stylet end, the distance travelled by the tube tip significantly increased as the bending angle increased. Mathematical analysis revealed that the stylet should be diagonally extracted (in the sagittal plane) at an appropriate angle, rather than along a straight path towards the direction of the stylet end. In simulated tracheal intubation, extraction force and force applied to the vocal cords both significantly increased as the bending angle increased. Compared with the 'hockey stick'-shaped stylet, the arcuate-shaped stylet resulted in reduced force. Our results indicate the potential risk for vocal cord injury when using hockey stick-shaped stylets with large bending angles.

The effects of Patent Blue dye on peripheral and cerebral oxyhaemoglobin saturations.

We measured the effect of Patent Blue dye on oxyhaemoglobin saturations after injection into breast tissue: 40 women had anaesthesia for breast surgery maintained with sevoflurane or propofol (20 randomly allocated to each). Saturations were recorded with a digital pulse oximeter, in arterial blood samples and with a cerebral tissue oximeter before dye injection and 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min afterwards. Patent Blue did not decrease arterial blood oxyhaemoglobin saturation, but it did reduce mean (SD) digital and cerebral oxyhaemoglobin saturations by 1.1 (1.1) % and 6.8 (7.0) %, p < 0.0001 for both. The falsely reduced oximeter readings persisted for at least 2 h. The mean (SD) intra-operative digital pulse oxyhaemoglobin readings were lower with sevoflurane than propofol, 97.8 (1.2) % and 98.8 (1.0) %, respectively, p < 0.0001.

Assessment of the potential for pathogen dispersal during high-flow nasal therapy.

High-flow nasal therapy is increasingly used in hospitals because of its effectiveness and patient comfort. However, pathogens in the patient's nasal and oral cavities may be dispersed by forced air. This study aimed to investigate the risk of pathogen dispersal during high-flow nasal therapy. Liquid and bacterial dispersal were assessed via in-vitro experimental set-ups using a manikin. Thickened water or fresh yeast solution mimicked saliva and nasal mucus secretions. Dispersal was limited to the proximal area of the face and nasal cannula, suggesting that high-flow nasal therapy does not increase the risk of droplet and contact infection.

Absorption spectroscopy of gold nanoisland films: optical and structural characterization.

Nanoisland films prepared by annealing thin gold films at high temperatures were imaged using scanning electron microscopy (SEM) and atomic force microscopy, and optically characterized through absorption spectroscopy. Thin gold films of effective thicknesses 2, 5 and 7 nm annealed at 500, 700 and 900 degrees C were fabricated and studied experimentally. The measured absorption characteristics in support of theoretical calculations showed that the shapes of gold islands were partial spheres. The position of the peak absorption wavelength measured with s-polarized light or at normal incidence confirmed that the island shape grew from a near-hemisphere towards a sphere with increasing annealing temperature. The SEM images confirmed that the size of islands increased from 15 nm in diameter to 40 nm in diameter as film thickness increased from 2 to 5 nm. The affect of the index of the substrate material on absorption characteristics were also studied by comparing the absorption spectra of gold island films on quartz and LaSF15 glass substrates. The use of gold nanoisland films for preparing localized surface plasmon resonance substrates was suggested as they held advantages over the gold colloid films.

Translocator protein 18kDa antagonist ameliorates stress-induced stool abnormality and abdominal pain in rodent stress models.

BACKGROUND: Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder characterized by abdominal pain and abnormal bowel habits, both of which are exacerbated by psychological stress. The translocator protein 18kDa (TSPO) is a marker of reactive gliosis in a number of central nervous system (CNS) diseases and responsible for many cellular functions, including neurosteroidogenesis. Although it has been reported that psychological stress disturbs neurosteroids levels, the pathophysiological relevance of TSPO in IBS is poorly understood. METHODS: We examined the effects of a TSPO antagonist, ONO-2952, on stress-induced stool abnormality and abdominal pain in rats, and on anxiety-related behavior induced by cholecystokinin. KEY RESULTS: Oral administration of ONO-2952 attenuated stress-induced defecation and rectal hyperalgesia in rats with an efficacy equivalent to that of a 5-HT3 receptor antagonist. In addition, ONO-2952 suppressed cholecystokinin-induced anxiety-like behavior with an efficacy equivalent to that of psychotropic drugs. On the other hand, ONO-2952 did not affect spontaneous defecation, gastrointestinal transit, visceral nociceptive threshold, and neurosteroid production in non-stressed rats even at a dose 10 times higher than its effective dose in the stress models. CONCLUSIONS AND INFERENCES: These results suggest that TSPO antagonism results in antistress action, and that ONO-2952 is a promising candidate for IBS without side effects associated with current treatment.

Diagnosis and treatment of obscure gastrointestinal bleeding using combined capsule endoscopy and double balloon endoscopy: 1-year follow-up study.

BACKGROUND AND STUDY AIMS: Several studies have shown the value of capsule endoscopy and double balloon endoscopy (DBE) in small-intestinal bleeding. The purpose of this study was to evaluate the impact of capsule endoscopy results on subsequent DBE examination, and the 1-year clinical outcome of this combined approach in patients with obscure gastrointestinal bleeding (OGIB). PATIENTS AND METHODS: A total of 45 consecutive patients with OGIB underwent capsule endoscopy. Patients with positive capsule endoscopy results underwent DBE for biopsy or therapy, and those with negative results underwent further assessment for possible diagnostic misses on capsule endoscopy. Tumors, ulcerations, and vascular lesions were considered as sources of bleeding. Diagnoses of OGIB lesions and clinical outcome were assessed 1 year after these examinations. RESULTS: Responsible lesions were found in 22 patients (49 %): 19 lesions in 18/45 patients (40 %) undergoing capsule endoscopy, and 18/36 patients (50 %) undergoing subsequent DBE. In all, 10 tumors, nine vascular lesions, and four ulcerations were found. In two patients, vascular lesions were only later diagnosed by conventional methods (4 %). Capsule endoscopy results guided our choice of the proper DBE model for successful therapeutic intervention in five patients. Re-bleeding rates were low during 1-year follow-up of the entire group (mean follow-up, 18.8 months): 5 % in cases with positive diagnoses on capsule endoscopy and/or DBE, and 12 % in negative cases. CONCLUSIONS: A combined approach using capsule endoscopy followed by DBE proves valuable in the diagnosis and treatment of patients with OGIB, leaves a low rate of undiagnosed bleeding sources, and has a good long-term outcome.

The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae.

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.

Effect of common driving sources to the feedback analysis of heart rate variability.

OBJECTIVES: This paper examines the operational characteristics of the multivariate autoregressive analysis applied to the simultaneous recordings of the instantaneous heart rate (IHR) and the change in systolic blood pressure (SBP). METHODS: The multivariate autoregressive model has been utilized to reveal the feedback characteristics between IHR and SBP. The model assumes the presence of independent set of driving forces to activate the system. However, it is likely that the driving forces may have correlation due to the presence of a common fluctuation source. This paper examines the effect of the presence of correlated components in the driving forces to the estimation accuracy of impulse responses characterizing the feedback properties. The two-dimensional autoregressive model driven by two correlated 1/f noises was chosen for the analysis of operational characteristics. The driving force was generated by a moving average system which simulates non-integer order integration. RESULTS: Computer simulation revealed that the mean square estimation errors of impulse responses sharply increase as relative power of common driving force exceeds 50%. However, the estimation accuracy and bias are found to be in permissible range in practice. CONCLUSIONS: These findings ensure the practical validity of utilizing multivariate autoregressive models for the feedback analysis between IHR and SBP where both signals have the common driving force.

Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity.

Recent work on isolated sinoatrial node cells from rabbit has suggested that sarcoplasmic reticulum Ca2+ release plays a dominant role in the pacemaker potential, and ryanodine at a high concentration (30 micromol/L blocks sarcoplasmic reticulum Ca2+ release) abolishes pacemaking and at a lower concentration abolishes the chronotropic effect of beta-adrenergic stimulation. The aim of the present study was to test this hypothesis in the intact sinoatrial node of the rabbit. Spontaneous activity and the pattern of activation were recorded using a grid of 120 pairs of extracellular electrodes. Ryanodine 30 micromol/L did not abolish spontaneous activity or shift the position of the leading pacemaker site, although it slowed the spontaneous rate by 18.9+/-2.5% (n=6). After ryanodine treatment, beta-adrenergic stimulation still resulted in a substantial chronotropic effect (0.3 micromol/L isoproterenol increased spontaneous rate by 52.6+/-10.5%, n=5). In isolated sinoatrial node cells from rabbit, 30 micromol/L ryanodine slowed spontaneous rate by 21.5+/-2.6% (n=13). It is concluded that sarcoplasmic reticulum Ca2+ release does not play a dominating role in pacemaking in the sinoatrial node. The full text of this article is available at http://www.circresaha.org.

Phosphorylation of elongation factor 2 in normal and malignant rat glial cells.

Certain calmodulin (CaM)-dependent protein kinases phosphorylate substrates have been implicated in regulating cellular proliferation. In this study, CaM-dependent phosphorylation has been examined in normal and tumor tissue from rat brain to determine whether differences exist. Using in vitro phosphorylation reactions, we compared endogenous substrates for Ca2+/CaM-dependent protein kinases in rat brain white matter (RBWM), a tissue rich in normal glia, to those of C6 rat glioma cells. A major phosphoprotein having a M(r) of 100,000 was observed in proliferating C6 cells that was not present in RBWM or in nonproliferating cells. Phosphorylation was stimulated by Ca2+ and CaM and inhibited by trifluoperazine. An antibody to elongation factor 2 (EF-2) immunoprecipitated the M(r) 100,000 protein from C6 cells. EF-2 was present in RBWM but was not phosphorylated. Homogenates of RBWM did not phosphorylate exogenous EF-2, which suggested the absence of CaM kinase III activity in normal glial tissue. Furthermore, the addition of purified, exogenous CaM kinase III to homogenates of RBWM resulted in EF-2 phosphorylation. These data demonstrate that a basal level of EF-2 phosphorylation exists in proliferating glioma cells that is markedly diminished or absent in normal glial tissue and is due to the activity of CaM kinase III.

Comparative study of 27-gauge vs 25-gauge vitrectomy for epiretinal membrane.

PURPOSE: The purpose of this study was to compare 27-gauge (27G) with 25-gauge (25G) microincision vitrectomy in patients with epiretinal membrane (ERM).ParticipantsSeventy-four eyes of 66 patients undergoing 3-port pars plana vitrectomy using 27G or 25G instrumentation. METHODS: Seventy-four eyes of 66 patients with ERM, who underwent 27G or 25G microincision vitrectomy were prospectively evaluated. RESULTS: The mean operation time for vitrectomy was significantly longer in the 27G group than in the 25G group (9.9±3.5 vs 6.2±2.7 min, respectively, P<0.0001). No statistically significant difference was found between the two groups in terms of the mean operation time for ERM-inner limiting membrane peeling (27G vs 25G: 20.2±9.9 vs 16.1±9.3 min, P=0.14), although the time for vitreous cutting was longer in the 27G group (9.9±3.5 vs 6.2±2.7 min, respectively, P<0.0001). The flare value, intraocular pressure (IOP), and rate of hypotony 1 day after surgery did not differ between the 27G and 25G groups (flare value: 18.7 vs 17.2; IOP: 8.8 vs 9.7 mm Hg; rate of hypotony: 30 vs 35%, respectively). There was no significant difference in the surgically induced astigmatism between the two groups in the follow-up period. The mean time required for wound closure did not show a significant difference between the 27G and 25G groups (7.7 vs 8.6 weeks, respectively). CONCLUSION: The 27G system is as safe and useful for ERM vitrectomy as the 25G system. Based on its potential, further improvement of 27G instruments could result in greater efficiency.

Effects of cholesterol evulsion on susceptibility to perfringolysin O of human erythrocytes.

Human erythrocytes preincubated with a phosphatidylcholine suspension (preincubated cells) showed decreased susceptibility to perfringolysin O, the decrease being strongly affected by preincubation time and temperature, and the phosphatidyl choline concentration. The binding of the toxin to the preincubated cells also decreased with the preincubation time and reached minimum at 37 degrees C for 6 h. Through this preincubation, about 30% of cholesterol was removed from cells without lysis. The susceptibility of preincubated cells to the toxin seemed to be affected by the amount of cholesterol removed from cells, but not by the cholesterol content of cell membranes. This indicates that most of the cholesterol interactive with the toxin is removable from cell membranes by preincubation with phosphatidylcholine suspension, and that the residual cholesterol is firmly constituted in the membrane structure and cannot interact with the toxin. After cholesterol evulsion by the preincubated plasma method (Murphy, J.R. (1962) J. Lab. Clin. Med. 60, 86-109 and 60, 571-578), cells also exhibited lower susceptibility to the toxin and to saponins, but higher susceptibility to lysophosphatidylcholine.

Differential stimulation by polyamines of phage DNA-directed in vitro synthesis of proteins.

The effect of polyamines on T7- and lambda rifd18 DNA-directed synthesis of proteins in an Escherichia coli cell-free system has been studied. When T7 DNA was used as a template, the degree of stimulation by spermidine of protein synthesis was larger with T7 RNA polymerase than with Mr 42 K protein, while the synthesis of Mr 13.5 K protein was not stimulated significantly by spermidine. The synthesis of T7 RNA polymerase was stimulated approx. 10-fold by 1 mM spermidine. When lambda rifd18 DNA was used as a template, the synthesis of beta beta' subunits of RNA polymerase was stimulated greatly by spermidine, while the synthesis of elongation factor Tu and ribosomal proteins was not stimulated significantly by spermidine. Spermidine stimulation of T7 DNA-directed synthesis of T7 RNA polymerase was at the level of both translation and transcription. The degree of stimulation by spermidine was greater at the level of translation. Putrescine stimulated the synthesis of T7 RNA polymerase and Mr 42 K protein to a small degree at the level of translation.

Alteration of human erythrocyte plasma membranes by perfringolysin O as revealed by freeze-fracture electron microscopy. Studies on Clostridium perfringens exotoxins V.

When human erythrocyte membranes were treated with perfringolysin O (Clostridium perfringens theta-toxin) and examined by electron microscopy after freeze-fracture, two ultrastructural alterations were observed in fracture faces of membrane. (1) A random aggregation of intramembranous particles was seen in the fracture face of the protoplasmic half (PF face) of all membranes treated with the toxin, even if at a low concentration (40 hemolytic units/ml). On the other hand, the aggregation in the fracture face of the exoplasmic half (EF face) was observed only in membranes treated with a high concentration (3300 hemolytic units/ml) for 2 h. (2) Round protrusions and "cavities" with 30 nm in diameter were visible in EF and PF faces of membranes treated with a high concentration, respectively. These structures were always protruded toward cytoplasmic side, but did not appear to form holes through the membrane. Ring and arc shaped structures with a dark center of 26 nm and a distinct border of 5 nm in width were observed when the toxin alone was negatively stained at a very high concentration (170,000 hemolytic units/ml). These structures were also produced in the presence of cholesterol even if the toxin concentration was low.

Membrane disorganization induced by perfringolysin O (theta-toxin) of Clostridium perfringens--effect of toxin binding and self-assembly on liposomes.

theta-Toxin (perfringolysin O) of Clostridium perfringens binds to membrane cholesterol with high (Kd approximately 10(-9) M) and low (Kd approximately 10(-7) M) affinities and causes membrane lysis of intact cells and liposomes. In order to understand the lytic process at the molecular level, the lysis of liposomes was investigated in comparison with that of intact cells. The toxin dose required to cause 50% lysis (RD50) of phosphatidylcholine/phosphatidylglycerol (82:18, mol/mol) liposomes containing 36-40 mol% cholesterol was 300-1400-times higher than the RD50 value for sheep or human erythrocytes when samples with the same cholesterol concentration were compared. However, the average number of toxin molecules bound per liposome vesicle at 50% lysis was estimated as 10-18 from the RD50 values, close to the number on erythrocytes at 50% lysis, suggesting that the number of toxin molecules adsorbed per vesicle is important for lysis. As to the toxin dose required for membrane lysis, no significant difference was observed between liposomes containing both high- and low-affinity toxin-binding sites and those containing only low-affinity sites, suggesting that theta-toxin molecules bound to low-affinity sites can assemble and cause membrane lysis as well as those bound to high-affinity sites. theta-Toxin assembles on liposomal membranes, as on erythrocytes, in a high-molecular-weight polymeric form as judged from sedimentation patterns in sucrose density-gradient centrifugation. The high-molecular-weight polymers were detected only under conditions where cell or liposome lysis occurred. At low toxin doses, slower sedimenting toxin oligomers and monomers were predominant on liposomal membranes. These results indicate that toxin assembly on membranes is essential for liposome lysis as it is for cell lysis and that assembly occurs on membranes without membrane proteins.

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate.

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.

Purification and properties of fMet-tRNAf deacylase from Escherichia coli.

A formylmethionyl-tRNAf deacylase has been purified about 330-fold from a crude initiation factor preparation (1 M NH4Cl ribosomal wash) from Escherichia coli Q13. The enzyme was nearly homogeneous and had an apparent molecular weight of 24 000. Rat liver methionyl-tRNAf and E. coli methionyl-tRNAm were not hydrolyzed significantly by the enzyme under standard conditions. Q beta RNA- and AUG(A)n-directed polypeptide synthesis was inhibited by the enzyme. The inhibition was at the level of initiation of polypeptide synthesis. The enzymatic activity was inhibited by various factors necessary for polypeptide synthesis. The activity was inhibited more by NH4Cl and spermidine than by Mg2+, GTP and ATP. The complex of formylmethionyl-tRNAf, initiation factor 2 and GTP was resistant to enzymatic hydrolysis, and the resistance was enhanced by the addition of AUG and ribosomes to the above reaction mixture.

A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.

An ARL1 mutation affected autophagic cell death in yeast, causing a defect in central vacuole formation.

When the cdc28 strain of Saccharomyces cerevisiae is incubated at restrictive temperatures, the yeasts digest themselves in 7 days by activating autophagic machinery. In parallel, the cell-proliferative activity decreases progressively after about 48 h. We have previously referred to this phenomenon as autophagic death. In the present study, we isolated and characterized a recessive mutant strain, dlp2, which delays the progression toward autophagic death. The cdc28 dlp2 cells contain many small vesicles instead of the large central vacuoles that are usually found in parental cdc28 cells. We showed that the dlp2 phenotype results from the presence of a single mutation in the gene ARL1 (ADP-ribosylation factor-like protein 1). Morphological and biochemical analyses of cdc28 dlp2 suggested that a defect in central vacuole formation is caused by aberrant membrane trafficking, although the protein-sorting to vacuoles is not affected. After a shift to a restrictive temperature, the components of the cytoplasm and nucleus of cdc28 dlp2 were condensed, with an accompanying formation of vesicles in the periphery (epiplasm) of the cells rather than an activation of the autophagic machinery. Introducing this ARL1 mutation into the normal ARL1 locus of the wild-type W303 strain again inhibited the progression of apoptotic cell death due to a defect in vacuole formation, which in this case was induced by the proapoptotic protein Bax. Thus, the ARL1 gene plays an important role in the formation of central vacuoles and in the progression of programmed cell death induced by cell-cycle arrest or Bax. These results suggested the presence of a programmed-cell death machinery in yeast that is similar to that related to the Type II cell death of mammalian cells characterized by autophagocytosis.