Reagent-free continuous thermal tert-butyl ester deprotection.

Continuous processing enables the use of non-standard reaction conditions such as high temperatures and pressures while in the liquid phase. This expands the chemist's toolbox and can enable previously unthinkable chemistry to proceed with ease. For a series of amphoteric amino acid derivatives, we have demonstrated the ability to hydrolyze the tert-butyl ester functionality in protic solvent systems. Using a continuous plug flow reactor at 120-240°C and 15-40min reaction times, no pH modification or additional reagents are needed to achieve the desired transformation. The method was then expanded to encompass a variety of more challenging substrates to test selectivity and racemization potential. The acid products were generally isolated as crystalline solids by simple solvent exchange after the deprotection reaction in good to high yield and purity.

Kilogram-scale prexasertib monolactate monohydrate synthesis under continuous-flow CGMP conditions.

Advances in drug potency and tailored therapeutics are promoting pharmaceutical manufacturing to transition from a traditional batch paradigm to more flexible continuous processing. Here we report the development of a multistep continuous-flow CGMP (current good manufacturing practices) process that produced 24 kilograms of prexasertib monolactate monohydrate suitable for use in human clinical trials. Eight continuous unit operations were conducted to produce the target at roughly 3 kilograms per day using small continuous reactors, extractors, evaporators, crystallizers, and filters in laboratory fume hoods. Success was enabled by advances in chemistry, engineering, analytical science, process modeling, and equipment design. Substantial technical and business drivers were identified, which merited the continuous process. The continuous process afforded improved performance and safety relative to batch processes and also improved containment of a highly potent compound.

Tissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infection.

BACKGROUND: Chlamydophila pneumoniae infection has been implicated as a potential risk factor for atherosclerosis, however the mechanism leading to persistent infection and its role in the disease process remains to be elucidated. METHODS: We validated the use of tissue microarray (TMA) technology, in combination with immunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raised against differentially expressed proteins under an interferon-gamma (IFN-gamma) induced model of chlamydial persistence. RESULTS: In the cell pellet array, we were able to identify differences in protein expression patterns between untreated and IFN-gamma treated samples. Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples. The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis. Subsequently, it was shown in a second TMA including archival atheromatous heart tissues from 12 patients undergoing heart transplantation, that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well, and were detectable morphologically within lesions by IHC. CONCLUSION: TMA technology proved useful in documenting functional proteomics data with the morphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may be useful markers for the detection of persistent infection in vitro and in vivo.

The use of heteroduplex analysis of polymerase chain reaction products to support the possible transmission of Legionella pneumophila from a malfunctioning automobile air conditioner.

Air conditioner condensates have not been previously associated with cases of Legionnaires' disease. We report the possible transmission of Legionella pneumophila serogroup 1 from a malfunctioning automobile air conditioning system's leaking water onto the floorboard of a car driven for a long distance by the patient. Heteroduplex analysis of polymerase chain reaction products was used to help establish an epidemiologic link between the water specimen and the patient.

Inhibition of oxidative burst and chemotaxis in human phagocytes by Legionella pneumophila zinc metalloprotease.

Legionella pneumophila produces several extracellular proteins, but their role in the pathogenesis of Legionnaires' disease is unclear. This study examined the effects of the L. pneumophila major secretory protein (Msp), a zinc metalloprotease, on the oxidative burst and chemotaxis of human phagocytes. Polymorphonuclear leucocytes (PMNLs) and adherent monocytes treated with sublethal amounts of Msp protease were stimulated with formyl-leucyl-methionyl-phenylalanine (fMLP) and opsonised zymosan particles (ZAP). A dose-dependent inhibition in superoxide anion production in response to both stimuli was seen, and complete inhibition was achieved in PMNLs and monocytes treated with Msp at concentrations of 1500 and 1000 U/ml, respectively. ZAP-induced chemiluminescence by PMNLs and mononuclear cells and fMLP-induced PMNL nitroblue tetrazolium dye reduction were both significantly inhibited. The chemotactic response of PMNLs to fMLP was inhibited in a dose-dependent manner and substantial inhibition (11% of control) was achieved with Msp 1200 U/ml. These results suggest that the L. pneumophila Msp protease alters human phagocyte functional responses significantly and may contribute to the pathogenesis of Legionnaires' disease.

The effects of pharmaceutical consumption and obesity on the quality of life in the organization of economic cooperation and development (OECD) countries.

OBJECTIVE: The objective of this study is to validate our earlier work on life expectancy with more recent data and, more importantly, to extend it to examine quality of life, not only the length of life. DESIGN AND SETTING: The analysis focuses on the production of health, disaggregating healthcare into pharmaceutical consumption and other healthcare. Going beyond our earlier work, measures of health include life expectancy and disability-adjusted life expectancy (DALE). Also, we consider the impact of obesity. The sample was 18 Organization of Economic Cooperation and Development (OECD) countries. The measure of pharmaceutical consumption is the best that is available for these countries. MAIN OUTCOME MEASURES AND RESULTS: Confirming our earlier work, pharmaceutical consumption has a positive and statistically significant effect on life expectancy at 40 and 60 years (significant at the 0.05 level, based on a two-tailed test). The effects are slightly larger than in the earlier work. Turning to DALE, pharmaceutical consumption has a positive and statistically significant effect at birth and at 60 years (significant at the 0.05 and 0.01 levels, respectively), based on a two-tailed test. The effects on DALE are larger than the effects on life expectancy. CONCLUSIONS: Increased pharmaceutical consumption helps improve quality of life, as well as life expectancy.

Evidence for persistent Chlamydia pneumoniae infection of human coronary atheromas.

To date, structures representing developmental stages of Chlamydia pneumoniae, especially persistent forms of this intracellular bacteria, have not been described in human atherosclerotic tissues using specific antibody labeling and transmission electron microscopy. Staining of atherosclerotic tissue from five patients seeking heart transplantation with gold-labeled antibodies specific for up-regulated chlamydial heat shock proteins, GroEL and GroES, and visualisation via transmission electron microscopy revealed intracellular, atypical, round to oval structures of variable diameter. These structures resembled reticulate bodies of Chlamydia, were surrounded by membranes and were located within smooth muscle cells, macrophages or fibroblasts. By using double immunogold electron microscopy technique (GroEL and GroES in combination with chlamydial LPS/MOMP antibodies), we demonstrated these structures were of chlamydial origin. In the current study, we demonstrated the presence of aberrant bodies of C. pneumoniae in vivo in archival coronary atheromatous heart tissues by the immunogold electron microscopy technique.

Is the United States an outlier in health care and health outcomes? A preliminary analysis.

U.S. health care is often seen as an outlier, with high costs and only middling outcomes. This view implies a household production function for health, with both health care and lifestyle serving as inputs. Building on earlier work by Miller and Frech (2004), we make this argument explicit by estimating a production function from augmented OECD data. This allows us to determine whether the U.S. is literally an outlier; which turns on whether the United States is very far off the production surface. We find that the Unites States is somewhat less productive than the average OECD country, but that a substantial part of the observed difference results from poor lifestyle choices, particularly obesity.

Identification of Chlamydia pneumoniae proteins in the transition from reticulate to elementary body formation.

Chlamydia pneumoniae is an important human respiratory pathogen that is responsible for an estimated 10% of community-acquired pneumonia and 5% of bronchitis and sinusitis cases. We examined changes in global protein expression profiles associated with the redifferentiation of reticulate body (RB) to elementary body (EB) as C. pneumoniae cells progressed from 24 to 48 h postinfection in HEp2 cells. Proteins corresponding to those showing the greatest changes in abundance in the beginning of the RB to EB transition were then identified from purified EBs. Among the 300 spots recognized, 35 proteins that were expressed at sufficiently high levels were identified by mass spectrometry. We identified C. pneumoniae proteins that showed more than 2-fold increases in abundance in the early stages of RB to EB transition, including several associated with amino acid and cofactor biosynthesis (Ndk, TrxA, Adk, PyrH, and BirA), maintenance of cytoplasmic protein function (GroEL/ES, DnaK, DksA, GrpE, HtrA, ClpP, ClpB, and Map), modification of the bacterial cell surface (CrpA, OmpA, and OmcB), energy metabolism (Tal and Pyk), and the putative transcriptional regulator TctD. This study identified C. pneumoniae proteins involved in the process of redifferentiation into mature, infective EBs and indicates bacterial metabolic pathways that may be involved in this transition. The proteins involved in RB to EB transition are key to C. pneumoniae infection and are perhaps suitable targets for therapeutic intervention.

Protein expression profiles of Chlamydia pneumoniae in models of persistence versus those of heat shock stress response.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-gamma) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42 degrees C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.

Estimating the compensating differential for employer-provided health insurance.

The theory of wage differentials argues that workers must pay for employer-provided group health insurance coverage through lower wages or reductions in other fringe benefits. This paper uses data from the 1988-90 Consumer Expenditure Survey (CEX) to estimate the wage-health insurance trade-off for male workers between the ages of 25 and 55. A fixed-effects model, which takes advantage of the rotating panel design of the CEX, is used to control for unobservable worker characteristics that are positively related with all forms of compensation, including wage earnings and health insurance coverage. I find a compensating differential for health insurance equal to roughly 10 to 11 percent of wages. Some caution is advised here due to the fact that I was unable to control for other fringe benefits, the most important being paid vacation and sick leave.

Analysis of altered protein expression patterns of Chlamydia pneumoniae by an integrated proteome-works system.

We have identified, analyzed, and quantified differential protein expression profile of five C. pneumoniae proteins, Adk (adenylate kinase), AhpC (thiol-specific antioxidant), CrpA (15 KD cysteine rich protein), Map (methionine aminopeptidae), and Cpn0710 (hypothetical protein) under normal versus persistent growth conditions induced by interferon-gamma, at different time intervals of their replicative cycle by successfully employing the latest proteomic analysis tool, PDQuest 2-D analysis software. We have also determined that this software represents a reliable analytical tool for mapping protein expression patterns in C. pneumoniae.

Increases in c-Jun N-terminal kinase/stress-activated protein kinase and p38 activity in monocyte-derived macrophages following the uptake of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, infects and replicates within a variety of eukaryotic cells. The purpose of the current study was to examine host cell signaling events immediately following uptake and early in the endocytic process (less than 1 h) following the phagocytosis of L. pneumophila. This examination focused on the protein kinase signal pathways to identify any aberrant signal(s) induced by L. pneumophila within its host, as a means to alter the normal endocytic pathway. The mitogen-activated protein kinase cascades are of interest due to their involvement in cellular regulation. The experiments were carried out with monocyte-derived macrophages (MDMs). All three mitogen-activated protein kinase cascades were activated when MDMs were inoculated with either Legionella strain (wild-type strain AA100 or dotA mutant GL10) or an Escherichia coli control. Whereas the avirulent treatments, GL10 and E. coli, exhibited a leveling off or a return to near basal levels of phosphorylation/activity of c-Jun N-terminal kinase by 60 min, the virulent strain AA100 exhibited a significantly increased level of activity through 60 min that was greater than that seen in GL10 (P = 0.025) and E. coli (P = 0.014). A similar trend was seen with p38 phosphorylation. Phosphorylation of mitogen-activated protein/ERK kinase (MEK) was decreased in strain AA100 compared to E. coli. Inhibition of the activity of either the stress-activated protein kinase/c-Jun N-terminal kinase or p38 pathway significantly decreased the ability of legionellae to replicate intracellularly, suggesting the necessity of these two pathways in its intracellular survival and replication.

Detailed protocol for purification of Chlamydia pneumoniae elementary bodies.

A detailed protocol for the growth and harvest of purified elementary bodies of Chlamydia pneumoniae is presented. This procedure utilizes a flask-to-flask passage scheme designed to achieve high bacterial titers in a short period of time.

Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells.

Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-gamma)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [(35)S]methionine/cysteine under normal conditions or IFN-gamma-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-gamma per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-gamma treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-gamma while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-gamma pressure to obtain a finer representation of the gene products involved in establishing persistence.

Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells.

The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-alpha). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-alpha, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-alpha-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.

Effect of macrolide antibiotics on human endothelial cells activated by Chlamydia pneumoniae infection and tumor necrosis factor-alpha.

This study investigated the potential anti-inflammatory activity of 3 macrolide antibiotics, clarithromycin, roxithromycin, and azithromycin, in an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVECs) were seeded in Transwell inserts, treated with serial dilutions of the antibiotics, and infected with Chlamydia pneumoniae or stimulated with tumor necrosis factor (TNF)-alpha. In HUVECs infected with C. pneumoniae or stimulated with TNF-alpha, both azithromycin and roxithromycin caused significant decreases in neutrophil and monocyte TEM, compared with antibiotic-free controls. Clarithromycin had no detectable effect in either group. Azithromycin caused significant decreases in interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1, whereas roxithromycin significantly decreased IL-8. This study indicates heterogeneity in the anti-inflammatory activity of these antibiotics. Mechanisms of monocyte and neutrophil TEM inhibition by azithromycin and roxithromycin are unclear but may be partially due to inhibition of IL-8 and MCP-1 production.