Binding-induced stabilization and assembly of the phage P22 tail accessory factor gp4.

To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.

The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging.

Shigella flexneri temperate bacteriophage Sf6 is of interest in part because its prophage expresses the oac gene that alters the antigenic properties of the surface O-antigen polysaccharide of its host bacterium. We have determined the complete sequence of its 39,044 bp genome. The sequence shows that Sf6 is a member of the canonical lambdoid phage group, and like other phages of this type has a highly mosaic genome. It has chromosomal regions that encode proteins >80% identical with at least 15 different previously characterized lambdoid phages and prophages, but 43% of the genome, including the virion assembly genes, is homologous to the genome of one phage, HK620. An analysis of the nucleotide differences between Sf6 and HK620 indicates that even these similar regions are highly mosaic. This mosaicism suggests ways in which the virion structural proteins might interact with each other. The Sf6 early operons are arranged like a typical lambdoid phage, with "boundary sequences" often found between functional modules in the "metabolic" genome domain. By virtue of high degree of similarity in the encoding genes and their DNA target sites, we predict that the integrase, early transcription anti-terminator, CI and Cro repressors, and CII protein of Sf6 have DNA binding specificities very similar to the homologous proteins encoded by phages HK620, lambda, 434 and P22, respectively. The late operon contains two tRNA genes. The Sf6 terminase genes are unusual. Analysis of in vivo initiation of the DNA packaging series showed that the Sf6 apparatus that recognizes DNA for packaging appears to cleave DNA for initiation of packaging series at many sites within a large region of about 1800 bp that includes a possible pac site. This is unlike previously characterized phage packaging mechanisms.

Transoesophageal echocardiography during coronary artery bypass procedures: impact on surgical planning.

INTRODUCTION: Intraoperative transesophageal echocardiography (iTEE) is widely accepted and routinely used during heart valve surgery. However, the impact of iTEE among patients undergoing coronary artery bypass grafting (CABG) is less well documented. In this study, we aim to define the impact of iTEE in patients undergoing myocardial revascularization for severe coronary artery disease. METHODS: We analyzed clinical data and preoperative and intraoperative echocardiograms of all adults who underwent on pump coronary bypass and iTEE between January 2008 and December 2008. RESULTS: 521 patients (mean age 69±14 years) were studied. New prebypass findings were obtained in 82 (15.7%) patients: in 62 (11.9%) of these patients, this information changed the surgical plan. New postbypass findings were obtained in 8 patients (1.5%) and the surgical plan was altered in 4 patients (0.7%). CONCLUSIONS: Overall new findings were obtained in 90 patients (17.2%) and the surgical plan was altered in 66 patients (12.6%). These data support the routine use of iTEE among patients undergoing surgical myocardial revascularization.

Effect of levosimendan on ventriculo-arterial coupling in patients with ischemic cardiomyopathy.

BACKGROUND: Levosimendan, a novel calcium sensitizer, enhances myocardial contractility without affecting intracellular calcium concentration. It also dilates peripheral arterial vessels by acting on ATP-dependent K(+) channels. Ventriculo-arterial coupling, the relationship between myocardial contractility and the arterial system, describes the efficiency of the cardiovascular system by analysing the relationship between myocardial contractility expressed by ventricular elastance (E(es)) and arterial elastance (E(a)). The aim of this prospective clinical investigation was to evaluate the effects of levosimendan on ventriculo-arterial coupling in patients with ischemic cardiomyopathy. METHODS: Fifteen patients with stable angina and left ventricular dysfunction underwent elective coronary surgery. Before surgery started, ventriculo-arterial coupling and several variables of cardiovascular performance were assessed by invasive monitoring and transoesophageal echocardiography before and after administration of levosimendan (12 mug/kg bolus) in coronary patients under general anesthesia. RESULTS: The cardiac index and ejection fraction increased significantly [from 1.92 +/- 0.4 to 2.1 +/- 0.4 l/min/m(2) (P = 0.0004) and from 31% +/- 6 to 40% +/- 9 (P = 0.001), respectively], while mean arterial pressure and systemic vascular resistances decreased significantly [from 83 +/- 10 to 72 +/- 5 mmHg (P = 0.0016) and from 997 +/- 341 to 855 +/- 324 dyne s/cm(5) (P = 0.0002), respectively]. After administration of levosimendan, E(a) decreased significantly (from 4.3 +/- 1.8 to 3.2 +/- 1.3 mmHg/ml/m(2), P= 0.005), while E(es) significantly increased (from 2.8 +/- 1.6 to 4.4 +/- 2.3 mmHg/ml/m(2), P= 0.05); as a result, E(a)/E(es) decreased significantly (from 1.76 +/- 1 to 0.83 +/- 0.2, P= 0.002). CONCLUSION: Levosimendan improves ventriculo-arterial coupling and cardiovascular performance in coronary patients with left ventricular dysfunction by enhancing myocardial contractility and reducing arterial elastance.

Right ventricular failure: physiology and assessment.

Right ventricular function can be altered in several disease states involving lungs and heart. Severe right ventricular dysfunction is a major determinant of outcome in such situations, and may strongly influence clinical management. The complex geometry of the right ventricle and the different physiology with respect to the left ventricle make the right ventricular failure difficult to define and assess. The response to increased afterload is the main determinant of right ventricle physiology in pathologic conditions. This consists of right ventricular hypertrophy and enlargement, with reduced coronary blood flow to the right ventricular wall, dilation of tricuspid annulus and displacement of interventricular septum. This latter change involves the left ventricular diastolic function, which is reduced by leftward septal shifting. In right ventricle myocardial ischemia and infarction the primum movens of altered right ventricular function is not an increase in afterload, but the ischemic involvement of the right ventricle, more often in the setting of an inferior acute myocardial infarction. The assessment of right ventricular failure is based on thermodilution by pulmonary artery catheter, contrast and radionuclide ventriculography, echocardiography, and magnetic resonance. Among these techniques, thermodilution and echocardiography play a relevant role in clinical scenarios, being readily available and feasible bedside.

Target controlled infusion: TCI.

Progress in computing technology has allowed the development of target controlled infusion devices, with drugs delivered to achieve specific predicted target blood drug concentrations. Target controlled infusion (TCI) system has been developed as a standardised infusion system for the administration of opioids, propofol and other anaesthetics by target controlled infusion. A set of pharmacokinetic parameters has been selected using computer simulation of a known infusion scheme. The selected model is incorporated into a computer-compatible infusion pump. Clinical trials with such systems have provided appropriate target concentrations for the administration of target controlled infusion of anaesthetic drugs. The technique of TCI strongly influences the development of intravenous anaesthesia and opens a scenario of new and exciting applications in peri-operative anaesthetic management. The launch of ''Diprifusor'' as the first commercially available TCI system for propofol was the cornerstone of a successful research period within the last decade, which evaluated the pharmacokinetic foundations of computer assisted intravenous drug delivery. Nowadays TCI technology is becoming a part of routine anaesthesia technique for the practitioner rather than a research tool for specialists and those who are enthusiasts of intravenous anaesthesia. Besides clinical application in anaesthesia, target controlled systems will play a significant role as research tools in the evaluation of drug interactions in anaesthesia and in the development of new control techniques for the administration of sedative and analgesic drugs in the peri-operative period.

Nucleotide sequence of the head assembly gene cluster of bacteriophage L and decoration protein characterization.

The temperate Salmonella enterica bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene, dec, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.

Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil.

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.

The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy.

The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.

Reduced compliance of left ventricle.

Diastolic function is essential for efficient systolic performance. A normal diastole allows left ventricle (LV) filling to occur under normal intracavitary pressure. It is an energy dependent process, as such affected by ischemia. Several factors influence diastolic function of the LV: the mitral valve area, the gradient between atrium and ventricle, LV relaxation and compliance, atrial compliance, the presence of sinus rhythm, the end-systolic volume. Echocardiography is the principal diagnostic tool to assess LV diastolic function noninvasively in clinical practice. Doppler evaluation allows to analyse each phase of LV diastole through measurement of transmitral and pulmonary veins flows velocities. Tissue Doppler echocardiography and color M-mode Doppler have also been introduced in the study of diastole. The use of echocardiography in the setting of diastolic dysfunction in ICU and operatory room has relevant implications in the management of haemodynamic instability, in vasoactive d rugs titration, in the detection of myocardial ischemia, and in performing prognostic stratification. These information can guide the management of cardiac patients undergoing cardiac and non cardiac surgery, in the perioperative phase, as well as in the management of critical ICU patients. On this basis evaluating the LV filling properties can contribute to improve the quality of treatments in such challenging situations.

Corrected sequence of the bacteriophage p22 genome.

We report the first accurate genome sequence for bacteriophage P22, correcting a 0.14% error rate in previously determined sequences. DNA sequencing technology is now good enough that genomes of important model systems like P22 can be sequenced with essentially 100% accuracy with minimal investment of time and resources.