SARS associated coronavirus has a recombinant polymerase and coronaviruses have a history of host-shifting.

The sudden appearance and potential lethality of severe acute respiratory syndrome associated coronavirus (SARS-CoV) in humans has focused attention on understanding its origins. Here, we assess phylogenetic relationships for the SARS-CoV lineage as well as the history of host-species shifts for SARS-CoV and other coronaviruses. We used a Bayesian phylogenetic inference approach with sliding window analyses of three SARS-CoV proteins: RNA dependent RNA polymerase (RDRP), nucleocapsid (N) and spike (S). Conservation of RDRP allowed us to use a set of Arteriviridae taxa to root the Coronaviridae phylogeny. We found strong evidence for a recombination breakpoint within SARS-CoV RDRP, based on different, well supported trees for a 5' fragment (supporting SARS-CoV as sister to a clade including all other coronaviruses) and a 3' fragment (supporting SARS-CoV as sister to group three avian coronaviruses). These different topologies are statistically significant: the optimal 5' tree could be rejected for the 3' region, and the optimal 3' tree could be rejected for the 5' region. We did not find statistical evidence for recombination in analyses of N and S, as there is little signal to differentiate among alternative trees. Comparison of phylogenetic trees for 11 known host-species and 36 coronaviruses, representing coronavirus groups 1-3 and SARS-CoV, based on N showed statistical incongruence indicating multiple host-species shifts for coronaviruses. Inference of host-species associations is highly sensitive to sampling and must be considered cautiously. However, current sampling suggests host-species shifts between mouse and rat, chicken and turkey, mammals and manx shearwater, and humans and other mammals. The sister relationship between avian coronaviruses and the 3' RDRP fragment of SARS-CoV suggests an additional host-species shift. Demonstration of recombination in the SARS-CoV lineage indicates its potential for rapid unpredictable change, a potentially important challenge for public health management and for drug and vaccine development.     

Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers

For the act of membrane fusion, there are two competing, mutually exclusive molecular models that differ in the structure of the initial pore, the pathway for ionic continuity between formerly separated volumes. Because biological “fusion pores” can be as small as ionic channels or gap junctions, one model posits a proteinaceous initial fusion pore. Because biological fusion pore conductance varies widely, another model proposes a lipidic initial pore. We have found pore opening and flickering during the fusion of protein-free phospholipid vesicles with planar phospholipid bilayers. Fusion pore formation appears to follow the coalescence of contacting monolayers to create a zone of hemifusion where continuity between the two adherent membranes is lipidic, but not aqueous. Hypotonic stress, causing tension in the vesicle membrane, promotes complete fusion. Pores closed soon after opening (flickering), and the distribution of fusion pore conductance appears similar to the distribution of initial fusion pores in biological fusion. Because small flickering pores can form in the absence of protein, the existence of small pores in biological fusion cannot be an argument in support of models based on proteinaceous pores. Rather, these results support the model of a lipidic fusion pore developing within a hemifused contact site.     

Intrathoracic pressure fluctuations move blood during CPR: Comparison of hemodynamic data with predictions from a mathematical model

Whether blood flow during cardiopulmonary resuscitation (CPR) results from intrathoracic pressure fluctuations or direct cardiac compression remains controversial. We developed a mathematical model that predicts that blood flow due to intrathoracic pressure fluctuations should be insensitive to compression rate over a wide range but dependent on the applied force and compression duration. If direct compression of the heart plays a major role, however, the model predicts that flow should be dependent on compression rate and force, but above a threshold, insensitive to compression duration. These differences in hemodynamics produced by changes in rate and duration form a basis for determining whether blood flow during CPR results from intrathoracic pressure fluctuations or from direct cardiac compression. The model was validated for direct cardiac compression by studying the hemodynamics of cyclic cardiac deformation following thoracotomy in four anesthetized, 21–32-kg dogs. As predicted by the model, there was no change in myocardial or cerebral perfusion pressures when the duration of compression was increased from 15% to 45% of the cycle at a constant rate of 60/min. There was, however, a significant increase in perfusion pressures when rate was increased from 60 to 150/min at a constant duration of 45%. The model was validated for intrathoracic pressure changes by studying the hemodynamics produced by a thoracic vest (vest CPR) in eight dogs. The vest contained a bladder that was inflated and deflated. Vest CPR changed intrathoracic pressure without direct cardiac compression, since sternal displacement was <0.8 cm. As predicted by the model and opposite to direct cardiac compression, there was no change in perfusion pressures when the rate was increased from 60 to 150/min at a constant duration of 45% of the cycle. Manual CPR was then studied in eight dogs. There was no surgical manipulation of the chest. Myocardial and cerebral blood flows were determined with radioactive microspheres and behaved as predicted from the model of intrathoracic pressure, not direct cardiac compression. At nearly constant peak sternal force (378–426 N), flow was significantly increased when the duration of compression was increased from short (13%–19% of the cycle) to long (40%–47%), at a rate of 60/min. Flow was unchanged, however, for an increase in rate from 60 to 150/min at constant compression duration. In addition, myocardial and cerebral flow correlated with their respective perfusion pressures. Thus vital organ perfusion pressures and flow for manual external chest compression are dependent on the duration of compression, but not on rates of compression of 60 and 150/min. These data are of course similar to those produced by vest CPR, where intrathoracic pressure is manipulated without sternal displacement, and to those predicted for movement of blood by intrathoracic pressure changes. These data are, however, opposite to those produced by cardiac deformation and to those predicted for movement blood by direct cardiac compression. We conclude that intrathoracic pressure fluctuations generate blood flow during manual CPR.     

Immunity after treatment of human schistosomiasis: association between cellular responses and resistance to reinfection.

Previous studies have demonstrated the development of an age-dependent resistance to reinfection after chemotherapeutic cure of the helminthic parasite Schistosoma mansoni. Here we report on a longitudinal investigation of cell-mediated responses in infected individuals before and after treatment which was designed to outline those parameters important in mediating a protective response. A well-defined study group of 89 individuals with an age range of 9 to 35 years was selected from an area of high S. mansoni transmission in the Machakos district of Kenya. Peripheral blood mononuclear cell proliferation and cytokine production (interleukin-2 [IL-2], gamma interferon IL-5, IL-4, and tumor necrosis factor) in response to different crude life cycle-stage antigens of S. mansoni were assessed longitudinally in vitro before, 3 months after, and 1 year after treatment. Detailed statistical analyses of the results from this study have indicated a clear negative association between the proliferative responses to adult- and schistosomulum-stage antigens and subsequent reinfection intensity in older individuals (14 to 35 years) which was not present in the younger individuals (9 to 13 years). This association was significant even after the effects of age, sex, and exposure had been accounted for in multiple regression analyses. Cytokines were detected predominantly in response to adult worm and egg antigen extracts. An inverse association between the two cytokines gamma interferon and IL-5 was detected in response to all antigens at the three time points investigated, indicating cross-regulation in the production of these two mediators. Differences in antigen-specific cytokine levels between the two age groups were detected, with significantly higher IL-5 levels detected in the older (more resistant) age group. An inverse correlation between this cytokine and reinfection was detected but could not be dissociated from the effects of age and exposure in multiple regression analysis.     

Reliability and validity of the Biodex system 3 pro isokinetic dynamometer velocity,torque and position measurements

This study quantitatively assessed the mechanical reliability and validity of position, torque and velocity measurements of the Biodex System 3 isokinetic dynamometer. Trial-to-trial and day-to-day reliability were assessed during three trials on two separate days. To assess instrument validity, measurement of each variable using the Biodex System 3 dynamometer was compared to a criterion measure of position, torque and velocity. Position was assessed at 5° increments across the available range of motion of the dynamometer. Torque measures were assessed isometrically by hanging six different calibrated weights from the lever arm. Velocity was assessed (30°/s to 500°/s) across a 70° arc of motion by manually accelerating the weighted lever arm. With the exception of a systematic decrease in velocity at speeds of 300°/s and higher, the Biodex System 3 performed with acceptable mechanical reliability and validity on all variables tested.DisclosureThe Biodex dynamometer used for this investigation was donated to the laboratory by Biodex Medical Systems. The authors have no commercial or proprietary interest in this device.     

β-Adrenergic stimulation does not regulate Na pump function in voltage-clamped ventricular myocytes of the rat heart

Summary Na pump current was measured in rat ventricular myocytes to determine if -adrenergic stimulation can directly modulate Na,K-ATPase activity. Enzymatically-isolated heart cells were voltage-clamped with a single patch electrode and Na pump current was briefly activated by rapidly increasing extracellular [K⁺] from 0 to 15 mM for 3–5 s after other ionic currents were blocked or inactivated. The salt solution in the voltage-clamping electrode included (in mM): (1) 100 Na⁺, 10 EGTA, (2) 5 Na⁺, 10 EGTA, or (3) 100 Na⁺, 7.5 Ca²⁺, 10 EGTA (free [Ca²⁺]=480 nM). With all three electrode solutions, Na pump current was not significantly changed after 2–4 min in the presence of 10 M isoprenaline. -adrenergic pathways were still intact as evidenced by the two-fold increase in Cd²⁺-sensitive Ba²⁺ current through calcium channels that was observed in the presence of isoprenaline. Thus, -adrenergic stimulation does not appear to directly regulate Na,K-ATPase activity in rat ventricular myocytes.