Performance in population models for count data,part II: A new SAEM algorithm

Analysis of count data from clinical trials using mixed effect analysis has recently become widely used. However, algorithms available for the parameter estimation, including LAPLACE and Gaussian quadrature (GQ), are associated with certain limitations, including bias in parameter estimates and the long analysis runtime. The stochastic approximation expectation maximization (SAEM) algorithm has proven to be a very efficient and powerful tool in the analysis of continuous data. The aim of this study was to implement and investigate the performance of a new SAEM algorithm for application to count data. A new SAEM algorithm was implemented in MATLAB for estimation of both, parameters and the Fisher information matrix. Stochastic Monte Carlo simulations followed by re-estimation were performed according to scenarios used in previous studies (part I) to investigate properties of alternative algorithms (Plan et al., 2008, Abstr 1372 []). A single scenario was used to explore six probability distribution models. For parameter estimation, the relative bias was less than 0.92% and 4.13% for fixed and random effects, for all models studied including ones accounting for over- or under-dispersion. Empirical and estimated relative standard errors were similar, with distance between them being <1.7% for all explored scenarios. The longest CPU time was 95 s for parameter estimation and 56 s for SE estimation. The SAEM algorithm was extended for analysis of count data. It provides accurate estimates of both, parameters and standard errors. The estimation is significantly faster compared to LAPLACE and GQ. The algorithm is implemented in Monolix 3.1, (beta-version available in July 2009).     

ICPC-2--international classification of primary health care and its application in Croatian health care

A consensus reached by the medical profession, Croatian Institute of Health Insurance, Croatian Institute of Public Health, and Ministry of Health constitutes a solution to the problem of a data standard required in building an information system for primary health care. This consists of accepting ICPC-2 as a data standard for our Family Medicine, Pediatrics and Women's Health. The classification structure of the International Classification of Primary Care allows, recommends and urges that special codes be established by individual states or local authorities when registering patients' reasons for seeking medical aid or medical procedures. Namely, it urges the authorities to set the codes for such reasons about which a state or local agreement or determination has been made. This is the first public presentation of the proposal Croatia's Additions to the International Classification of Primary Care. They are essential to its implementation in our health insurance, health statistics and medical informatics.     

Recognizing, Scoring, and Predicting Blast Injuries RID="" ID="" This International Association for the Surgery of Trauma and Surgical Intensive Care (IATSIC) article was presented at the 37th World Congress of Surgery International Surgical Week (ISW97), Acapulco, Mexico, August 24–30, 1997.

n = 665, 51%) had an ISS ranging from 0 to 34 (mean 13) had wounds ranging from G1ST (soft tissue wounds caused by low energy transfer) to G3VF (massive wounds with fractures and injury of vital structures) according to the RCWC, with PSS/IS scores from 2 to 105 (mean 60). Statistically significant correlation was found between ISS and PSS/IS as well as RCWC and PSS/IS. Cytokines (IL-1, TNF _alpha ) and amino acids responded to a blast injury in similar manner as to gunshot wounds with a greater ISS or more severe RCWC injury type. The subjective sensations in blasted patients (deafness, thoracic pain, vertigo) and mediators, confirmed in previous experimental investigations as important factors in the pathogenesis of blast injuries (TxA ₂ , sulfidopeptide leukotrienes) were relationed only to the PSS/IS.RID=" ID=" <E5>Correspondence to:</E5> I. Cernak, M.D., Ph.D.     

Complete genome sequence of an M1 strain of Streptococcus pyogenes

The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes, a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial "molecular mimicry" of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.     

Clinical Significance of Laboratory-determined Aspirin Poor Responsiveness After Primary Percutaneous Coronary Intervention

Aims

The objective of the present substudy was to examine whether aspirin poor/high responsiveness (APR/AHR) is associated with increased rates of major adverse cardiovascular events (MACE) and serious bleeding after primary percutaneous coronary intervention (PPCI).

Methods

We analyzed 961 consecutive ST-elevation acute myocardial infarction patients who underwent PPCI between February 2008 and June 2011. Multiplate analyser (Dynabite, Munich, Germany) was used for the assessment of platelet reactivity. APR/AHR were defined as the upper/lower quintiles of ASPI values, determined 24 h after aspirin loading. APR patients were tailored using 300 mg maintenance dose for 30 days. The co-primary end points at 30 days were: MACE (death, non-fatal infarction, ischemia-driven target vessel revascularization and ischemic stroke) and serious bleeding according to the BARC classification.

Results

One hundred and 90 patients were classified as APR, and 193 patients as AHR. At admission, compared with aspirin sensitive patients (ASP), patients with APR had more frequently diabetes, anterior infarction and heart failure, while AHR patients had reduced values of creatine kinase, leukocytes, heart rate and systolic blood pressure. Compared with ASP, the rates of 30-day primary end points did not differ neither in APR group including tailored patients (MACE, adjusted OR 1.02, 95%CI 0.47-2.17; serious bleeding, adjusted OR 1.92, 95%CI 0.79-4.63), nor in patients with AHR (MACE, adjusted OR 1.58, 95%CI 0.71-5.51; serious bleeding, adjusted OR 0.69, 95%CI 0.22-2.12).

Conclusions

The majority of APR patients were suitable for tailoring. Neither APR including tailored patients nor AHR were associated with adverse 30-day efficacy or safety clinical outcomes.

     

ZnO Nanoparticles Impose a Panmetabolic Toxic Effect Along with Strong Necrosis,Inducing Activation of the Envelope Stress Response in Salmonella enterica Serovar Enteritidis

In this study, we tested the antimicrobial activity of three metal nanoparticles (NPs), ZnO, MgO, and CaO NPs, against Salmonella enterica serovar Enteritidis in liquid medium and on solid surfaces. Out of the three tested metal NPs, ZnO NPs exhibited the most significant antimicrobial effect both in liquid medium and when embedded on solid surfaces. Therefore, we focused on revealing the mechanisms of surface-associated ZnO biocidal activity. Using the global proteome approach, we report that a great majority (79%) of the altered proteins in biofilms formed by Salmonella enterica serovar Enteritidis were downregulated, whereas a much smaller fraction (21%) of proteins were upregulated. Intriguingly, all downregulated proteins were enzymes involved in a wide range of the central metabolic pathways, including translation; amino acid biosynthetic pathways; nucleobase, nucleoside, and nucleotide biosynthetic processes; ATP synthesis-coupled proton transport; the pentose phosphate shunt; and carboxylic acid metabolic processes, indicating that ZnO NPs exert a panmetabolic toxic effect on this prokaryotic organism. In addition to their panmetabolic toxicity, ZnO NPs induced profound changes in cell envelope morphology, imposing additional necrotic effects and triggering the envelope stress response of Salmonella serovar Enteritidis. The envelope stress response effect activated periplasmic chaperones and proteases, transenvelope complexes, and regulators, thereby facilitating protection of this prokaryotic organism against ZnO NPs.     

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein’s folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein’s functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.All proteins are biosynthesized as linear chains, and most need to fold into 3D structures to function. Studies on protein folding in buffers have revealed that a kinetic competition typically exists between protein folding, misfolding, and aggregation. It is the role of the protein homeostasis or proteostasis network in each subcellular compartment to regulate this competition and keep the folded and functional proteome within the physiological concentration range, while minimizing misfolding and aggregation in the face of stresses (1–4). It remains a challenge to discern how the proteostasis network affects the folding of proteins into biologically active conformations required for function in vivo (5).Current methodologies allow for quantification of the partitioning of a protein of interest (POI) between soluble and aggregated states but cannot determine the proportion of the soluble population that is properly folded and functional. Published folding probes have the potential to report on the folded fraction in cells or cell lysate (6–9); however, the extent to which they shift folding equilibria and quantify the folded and functional fraction faithfully has not been studied. Herein, we create POI folding probes by adapting the principle of activity-based protein profiling (10) to quantify the soluble folded and functional fraction of a particular protein in a cell lysate. We seek folding probes that bind to and selectively react with only the folded and functional state of a POI in a cell, leaving the nonfunctional states and other cellular proteins unmodified (Fig. 1A).Open in a separate windowFig. 1.A small molecule folding probe strategy to quantify the soluble folded and functional fraction of a POI in a cell lysate. (A) Overview of the general strategy to selectively covalently label a folded and functional POI without labeling its nonfunctional conformations and other cellular proteins. (B) The experimental scheme to quantify the ratio of the soluble POI that is functional (R_f).Fluorescent folding probes for the de novo-designed enzyme, retroaldolase (RA) (11), and fluorogenic folding probes (12) for the nonenzyme protein, transthyretin (TTR), were developed and scrutinized. We show that destabilized mutant RA and TTR proteins partition into folded and functional as well as misfolded soluble conformations and that this partitioning is sensitive to proteostasis network perturbations. Experiments show that a snapshot of the distribution between folded and functional vs. soluble and misfolded conformational states can be preserved during the small molecule folding probe labeling period, provided that the cellular chaperone holdase activity is sufficient, achieved by rapid ATP depletion in parallel with cell lysis. Sufficient chaperone/chaperonin holdase activity minimizes changes in the folded and functional concentration associated with probe binding and reaction with the POI and renders the relative folding and conjugation rates much less influential.