A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein.

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 µM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection. We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Utilización de la ventilación mecánica en 72 unidades de cuidados intensivos en España / Utilization of mechanical ventilation in 72 Spanish intensive care units

Fundamento. La ventilación mecánica es una técnica fundamental en las unidades de cuidados intensivos (UCI). El objetivo es conocer su utilización y las diferencias en su aplicación en 72 UCI españolas. Métodos. Estudio de cohortes de los pacientes ventilados durante más de 12 h. Se registraron datos demográficos, indicación de la ventilación mecánica, parámetros ventilatorios, modos de ventilación y desconexión, y días de soporte ventilatorio, días de estancia y situación al alta. Resultados. Se incluyó a 1.103 pacientes (29 per cent) de los ingresados, un 66 per cent de varones, con una mediana de edad de 65 años y del SAPS II de 43.No se observaron diferencias geográficas en la aplicación de la ventilación mecánica ni en los desenlaces principales. En el 64 per cent la indicación de ventilación fue insuficiencia respiratoria. Al inicio, la modalidad asistida-controlada fue la más utilizada (90 per cent), con un volumen tidal medio (desviación estándar [DE]) 8,9 (2,0) ml/kg y PEEP 5,5 (2,2) cmH2O. El 4 per cent recibió ventilación no invasora, el 68 per cent de los enfermos no precisó intubación. La duración de la ventilación fue 7 (8) días. El método de desconexión más utilizado fue una prueba única diaria de respiración espontánea (58 per cent). La duración fue de 3 (5) días. La incidencia de traqueostomía fue 15 per cent y se realizó a los 14 (8) días. La mortalidad en la UCI fue del 32,8 per cent y en el hospital del 42,8 per cent. Conclusiones. La comparación de la utilización de la ventilación mecánica en 72 UCI españolas evidencia una práctica similar, con mínimas diferencias en el uso de los modos de ventilación y desconexión y una similar mortalidad y días de estancia (AU)

Role of transesophageal echocardiography in the assessment of patients with blunt chest trauma: correlation of echocardiographic findings with the electrocardiogram and creatine kinase monoclonal antibody measurements.

OBJECTIVES: This study was designed to evaluate the usefulness of transesophogeal echocardiography (TEE) for detecting cardiac damage after blunt chest trauma (BCT). BACKGROUND: Multiple methods have been used to detect cardiac damage after a BCT, but none has been demonstrated to be sensitive, specific, and feasible enough. METHODS: This multicenter prospective trial was designed to evaluate the usefulness of TEE in the assessment of patients with BCT and to compare the TEE findings with those provided by the electrocardiogram (ECG) and cardiac isoenzymes assay. One hundred seventeen consecutive patients with a significant BCT were enrolled. A TEE was performed in each patient. Serial ECGs and plasma profiles of creatine kinase (CK) and CK-monoclonal antibody (MB) were obtained. RESULTS: Sixty-six (56%) patients had pathologic findings in the TEE attributed to the BCT (group A). In the remaining 51 (44%) patients the TEE was normal (group B). An abnormal ECG was more frequent in group A (59% vs 24%; p < 0.001), and the serum CK-MB peak level was also higher in group A (174 +/- 30 U/L vs 93 +/- 21 U/L; p = 0.05). Relative to pathologic TEE findings, the sensitivity and specificity of an abnormal ECG were 59% and 73% and of high CK-MB with CK-MB/CK > 5% were 64% and 52%, respectively. CONCLUSIONS: We conclude that TEE can be routinely and safely performed for diagnosing cardiac injuries after a BCT and plays an important role in the evaluation and treatment of these patients. EGG and CK-MB assay are not good methods for detecting cardiac damage in this setting.