The impact of comorbidities and their stacking on short- and long-term prognosis of patients over 50 with community-acquired pneumonia.

BACKGROUND: The prognosis of patients hospitalized with community-acquired pneumonia (CAP) with regards to intensive care unit (ICU) admission, short- and long-term mortality is correlated with patient's comorbidities. For patients hospitalized for CAP, including P-CAP, we assessed the prognostic impact of comorbidities known as at-risk (AR) or high-risk (HR) of pneumococcal CAP (P-CAP), and of the number of combined comorbidities. METHODS: Data on hospitalizations for CAP among the French 50+ population were extracted from the 2014 French Information Systems Medicalization Program (PMSI), an exhaustive national hospital discharge database maintained by the French Technical Agency of Information on Hospitalization (ATIH). Their admission diagnosis, comorbidities (nature, risk type and number), other characteristics, and their subsequent hospital stays within the year following their hospitalization for CAP were analyzed. Logistic regression models were used to assess the associations between ICU transfer, short- and 1-year in-hospital mortality and all covariates. RESULTS: From 182,858 patients, 149,555 patients aged ≥ 50 years (nonagenarians 17.8%) were hospitalized for CAP in 2014, including 8270 with P-CAP. Overall, 33.8% and 90.5% had ≥ 1 HR and ≥ 1 AR comorbidity, respectively. Cardiac diseases were the most frequent AR comorbidity (all CAP: 77.4%). Transfer in ICU occurred for 5.4% of CAP patients and 19.4% for P-CAP. Short-term and 1-year in-hospital mortality rates were 10.9% and 23% of CAP patients, respectively, significantly lower for P-CAP patients: 9.2% and 19.8% (HR 0.88 [95% CI 0.84-0.93], p < .0001). Both terms of mortality increased mostly with age, and with the number of comorbidities and combination of AR and HR comorbidities, in addition of specific comorbidities. CONCLUSIONS: Not only specific comorbidities, but also the number of combined comorbidities and the combination of AR and HR comorbidities may impact the outcome of hospitalized CAP and P-CAP patients.

Kinetics of myosin subfragment-1-induced condensation of G-actin into oligomers, precursors in the assembly of F-actin-S1. Role of the tightly bound metal ion and ATP hydrolysis.

In a low ionic strength buffer and in the absence of free ATP, the interaction of G-actin (G) with myosin subfragment-1 (S1) leads to the formation of arrowhead-decorated F-actin-S1 filaments, through a series of elementary steps. The initial formation of GS and G2S complexes is followed by their condensation into short oligomers. The kinetics of formation of G-actin-S1 oligomers have been monitored in a stopped-flow apparatus using a combination of light scattering and fluorescence of NBD-labeled actin. Oligomers appear more stable and are formed at a faster rate from MgATP-G-actin than from CaATP-G-actin. The actin-bound ATP is hydrolyzed when oligomers are formed from MgATP-G-actin, not when they are formed from CaATP-G-actin. The formation of oligomers is energetically favored in the presence of cytochalasin D. All data are consistent with the view that the actin-actin interactions which take place upon condensation of GS and G2S into oligomers are very similar to lateral actin-actin interactions along the short pitch helix of actin filaments, which are involved in actin nucleation. These interactions trigger ATP hydrolysis on actin.

Mechanism of myosin subfragment-1-induced assembly of CaG-actin and MgG-actin into F-actin-S1-decorated filaments.

The kinetics and mechanism of myosin subfragment-1-induced polymerization of G-actin into F-actin-S1-decorated filaments have been investigated in low ionic strength buffer and in the absence of free ATP. The mechanism of assembly of F-actin-S1 differs from salt-induced assembly of F-actin. Initial condensation of G-actin and S1 into oligomers in reversible equilibrium is a prerequisite step in the formation of F-actin-S1 . Oligomers have a relatively low stability (10(6) M-1) and contain S1 in a molar ratio to actin close to 0.5. Increased binding of S1 up to a 1:1 molar ratio to actin is associated with further irreversible condensation of oligomers into large F-actin-S1 structures of very high stability. In contrast to salt-induced assembly of F-actin, no monomer-polymer equilibrium, characterized by a critical concentration, can be defined for F-actin-S1 assembly, and end-to-end annealing of oligomers is predominant over growth from nuclei in the kinetics. Simultaneous recordings of the changes in light scattering, pyrenyl- and NBD-actin fluorescence, ATP hydrolysis, and release of Pi during the polymerization process have been analyzed to propose a minimum kinetic scheme for assembly, within which several elementary steps, following oligomer formation, are required for assembly of F-actin-S1. ATP hydrolysis occurs before polymerization of MgATP-G-actin but not of CaATP-G-actin. The release of inorganic phosphate occurs on F-actin-S1 at the same rate as on F-actin.

Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay.

ATP and GTP are hydrolyzed during self-assembly of actin and tubulin, respectively. It is known that nucleotide is hydrolyzed on the polymer in two consecutive steps, chemical cleavage of the gamma-phosphate followed by the slower release of Pi. This last step has been shown to play a crucial role in the dynamics of actin filaments and microtubules. Thus far, evidence for a transient GDP-Pi state in microtubule assembly has been obtained using a glass fiber filter assay that had a poor time resolution [Melki, R., Carlier, M.-F., & Pantaloni, D. (1990) Biochemistry 29, 8921-8932]. We have used a new Pi assay [Webb, M. R. (1992) Proc. natl. Acad. Sci. U.S.A. 89, 4884-4887], in which the purine phosphorylase catalyzes the phosphorolysis of 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) into mercaptopurine and ribose phosphate, which is accompanied by an increase in absorbance. This enzyme-linked assay has been used to follow the release of Pi during polymerization of Mg-actin. A value of 350 s was found for the half-time for Pi release on F-actin, in good agreement with previous determinations. The release of Pi following GTP hydrolysis in microtubule assembly was followed using a stopped-flow apparatus. Rapid microtubule assembly was achieved using taxol. The use of a stopped-flow apparatus permitted the continuous recording, with a dead time of 0.8 ms, of both time courses of microtubule assembly and Pi release with greatly improved time resolution. The release of Pi developed with a short lag (35 and 2 s for G-actin and tubulin, respectively) following assembly and appeared 50-fold faster on microtubules than on actin filaments.

Kinetics of the interaction of myosin subfragment-1 with G-actin. Effect of nucleotides and DNaseI.

The kinetics of interaction of monomeric pyrenyl-labeled G-actin with myosin subfragment-1 (S1 (A1) and S1(A2) isomers) has been examined in the stopped-flow at low ionic strength. The data confirm the previously reported existence of binary GS and ternary G2S complexes. The increase in pyrenyl-actin fluorescence which monitors the G-actin-S1 interactions is linked to the isomerization of these complexes following rapid equilibrium binding steps. The rates of isomerization are approximately 200 s-1 for GS and approximately 50 s-1 for G2S at 4 degrees C and in the absence of ATP. DNaseI and S1 bind G-actin essentially in a mutually exclusive fashion. Both GS and G2S are dissociated by MgATP and MgADP. The kinetics and mechanism of ATP-induced dissociation of G2S are quantitatively close to the ATP-induced dissociation of F-actin-S1, which indicates the G2S is a good model for the F-actin-S1 interface. GS and G2S display different kinetic behaviors in response to nucleotides, GS being less efficiently dissociated than G2S by MgATP. This result suggests that different mechanical properties of the cross-bridge might correlate with different orientations of the myosin head and different actin/myosin binding ratios.

Actin polymerization: regulation by divalent metal ion and nucleotide binding, ATP hydrolysis and binding of myosin.

Actin filaments are major dynamic components of the cytoskeleton of eukaryotic cells. Assembly of filaments from monomeric actin occurs with expenditure of energy, the tightly bound ATP being irreversibly hydrolyzed during polymerization. This dissipation of energy perturbs the laws of reversible helical polymerization defined by Oosawa and Asakura (1975), and affects the dynamics of actin filaments. We have shown that ATP hydrolysis destabilizes actin-actin interactions in the filament. The destabilization is linked to the liberation of Pi that follows cleavage of gamma-phosphate. Pi release therefore plays the role of a conformational switch. Because ATP hydrolysis is uncoupled from polymerization, the nucleotide content of the filaments changes during the polymerization process, and filaments grow with a stabilizing "cap" of terminal ADP-Pi subunits. The fact that the dynamic properties of F-actin are affected by ATP hydrolysis results in a non-linear dependence of the rate of filament elongation on monomer concentration. Possible modes of regulation of filament assembly may be anticipated from the basic properties of actin. We have shown that the tightly bound divalent metal ion (Ca2+ or Mg2+) interacts with the beta- and gamma-phosphates of ATP bound to actin, and that the Me-ATP bidentate chelate is bound to G-actin in the A configuration. The nature of the bound metal ion affects the conformation of actin and the rate of ATP hydrolysis. In motile living cells, a large pool of actin is maintained unpolymerized by interaction with G-actin binding proteins such as thymosin beta 4 and its variants or profilin. Part of this pool is released to increase the F-actin pool upon cell stimulation. The role of G-actin polymerizing proteins may be crucial in defining the patterns of filament assembly in these situations. The myosin head (myosin subfragment-1) may be considered as a model actin polymerizing protein, may be the closest model to the short tailed myosin I family. The mechanism of assembly of decorated filaments from G-actin and myosin subfragment-1 has therefore been examined.

Conformational changes in subdomain-2 of G-actin upon polymerization into F-actin and upon binding myosin subfragment-1.

The susceptibility of subdomain-2 of actin to different proteases has been examined, for G-actin, F-actin, G-actin-S1(A2) and F-actin-S1(A2) complexes on a comparative basis. The sites of subtilisin, alpha-chymotrypsin and trypsin attack, exposed on G-actin, are protected in F-actin, F-actin-S1(A2) as well as in the G-actin-S1(A2) complex. In contrast, a new cleavage site (Arg39-His40) for ArgC protease, which is protected in G-actin, is exposed in G-actin-S1(A2) as well as in F-actin and F-actin-S1(A2). These results are consistent with the previously proposed structural analogy between the ternary (G-actin)2S1 and the F-actin-S1 complexes, and provide information on the mechanism of S1-induced polymerization of G-actin.