Machine learning amplifies the effect of parental family history of Alzheimer's disease on list learning strategy.

Identification of preclinical Alzheimer's disease (AD) is an essential first step in developing interventions to prevent or delay disease onset. In this study, we examine the hypothesis that deeper analyses of traditional cognitive tests may be useful in identifying subtle but potentially important learning and memory differences in asymptomatic populations that differ in risk for developing Alzheimer's disease. Subjects included 879 asymptomatic higher-risk persons (middle-aged children of parents with AD) and 355 asymptotic lower-risk persons (middle-aged children of parents without AD). All were administered the Rey Auditory Verbal Learning Test at baseline. Using machine learning approaches, we constructed a new measure that exploited finer differences in memory strategy than previous work focused on serial position and subjective organization. The new measure, based on stochastic gradient descent, provides a greater degree of statistical separation (p = 1.44 × 10-5) than previously observed for asymptomatic family history and non-family history groups, while controlling for apolipoprotein epsilon 4, age, gender, and education level. The results of our machine learning approach support analyzing memory strategy in detail to probe potential disease onset. Such distinct differences may be exploited in asymptomatic middle-aged persons as a potential risk factor for AD.

Lentiviral-based BMP4 in vivo gene transfer strategy increases pull-out tensile strength without an improvement in the osteointegration of the tendon graft in a rat model of biceps tenodesis.

BACKGROUND: The present study aimed to develop a rat model of biceps tenodesis and to assess the feasibility of a lentiviral (LV)-based bone morphogenetic protein (BMP) 4 in vivo gene transfer strategy for healing of biceps tenodesis. METHODS: A rat model of biceps tenodesis was developed with an interference-fit open surgical technique. A LV vector expressing a BMP4 gene or ß-galactosidase (ß-gal) control gene was applied to the bone tunnel and the tendon graft before its insertion into the bone tunnel. Osteointegration was assessed by histology and pull-out tensile strength was measured by a biomechanical test suitable for small rat biceps tendon grafts. RESULTS: Neo-chondrogenesis was seen at the tendon-bone interface of LV-BMP4-treated but not control rats. The LV-BMP4-treated rats showed 32% (p < 0.05) more newly-formed trabecular bone at the tendon-bone junction than the LV-ß-gal-treated controls after 3 weeks. However, the sites of neo-chondrogenesis and new bone formation in the LV-BMP4-treated tenodesis were highly spotty. Although the LV-BMP4 strategy did not promote bony integration of the tendon graft, it yielded a 29.5 ± 11.8% (p = 0.066) increase in improvement the pull-out strength of rat biceps tendons compared to the LV-ß-gal treatment after 5 weeks. CONCLUSIONS: Although the LV-BMP4 in vivo gene transfer strategy did not enhance osteointegration of the tendon graft, it yielded a marked improvement in the return of the pull-out strength of the tendon graft. This presumably was largely a result of the bone formation effect of BMP4 that traps or anchors the tendon graft onto the bony tunnel.

Sleep-dependent improvement in visuomotor learning: a causal role for slow waves.

STUDY OBJECTIVES: Sleep after learning often benefits memory consolidation, but the underlying mechanisms remain unclear. In previous studies, we found that learning a visuomotor task is followed by an increase in sleep slow wave activity (SWA, the electroencephalographic [EEG] power density between 0.5 and 4.5 Hz during non-rapid eye movement sleep) over the right parietal cortex. The SWA increase correlates with the postsleep improvement in visuomotor performance, suggesting that SWA may be causally responsible for the consolidation of visuomotor learning. Here, we tested this hypothesis by studying the effects of slow wave deprivation (SWD). DESIGN: After learning the task, subjects went to sleep, and acoustic stimuli were timed either to suppress slow waves (SWD) or to interfere as little as possible with spontaneous slow waves (control acoustic stimulation, CAS). SETTING: Sound-attenuated research room. PARTICIPANTS: Healthy subjects (mean age 24.6 +/- 1.0 years; n = 9 for EEG analysis, n = 12 for behavior analysis; 3 women). MEASUREMENTS AND RESULTS: Sleep time and efficiency were not affected, whereas SWA and the number of slow waves decreased in SWD relative to CAS. Relative to the night before, visuomotor performance significantly improved in the CAS condition (+5.93% +/- 0.88%) but not in the SWD condition (-0.77% +/- 1.16%), and the direct CAS vs SWD comparison showed a significant difference (P = 0.0007, n = 12, paired t test). Changes in visuomotor performance after SWD were correlated with SWA changes over right parietal cortex but not with the number of arousals identified using clinically established criteria, nor with any sign of "EEG lightening" identified using a novel automatic method based on event-related spectral perturbation analysis. CONCLUSION: These results support a causal role for sleep slow waves in sleep-dependent improvement of visuomotor performance.

Statistical modeling of dental unit water bacterial test kit performance.

OBJECTIVE: While it is important to monitor dental water quality, it is unclear whether in-office test kits provide bacterial counts comparable to the gold standard method (R2A). Studies were conducted on specimens with known bacterial concentrations, and from dental units, to evaluate test kit accuracy across a range of bacterial types and loads. METHODOLOGY: Colony forming units (CFU) were counted for samples from each source, using R2A and two types of test kits, and conformity to Poisson distribution expectations was evaluated. Poisson regression was used to test for effects of source and device, and to estimate rate ratios for kits relative to R2A. RESULTS: For all devices, distributions were Poisson for low CFU/mL when only beige-pigmented bacteria were considered. For higher counts, R2A remained Poisson, but kits exhibited over-dispersion. Both kits undercounted relative to R2A, but the degree of undercounting was reasonably stable. Kits did not grow pink-pigmented bacteria from dental-unit water identified as Methylobacterium rhodesianum. CONCLUSION: Only one of the test kits provided results with adequate reliability at higher bacterial concentrations. Undercount bias could be estimated for this device and used to adjust test kit results. Insensitivity to methylobacteria spp. is problematic.

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF.

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.

Kinase drug discovery by affinity selection/mass spectrometry (ASMS): application to DNA damage checkpoint kinase Chk1.

Kinase enzymes are involved in a vast array of biological processes associated with human disease; therefore, selective kinase inhibition by small molecules and therapeutic antibodies is an area of intense study. The authors show that drug candidates with immediate value for biological preclinical evaluation can be identified directly through ultra-efficient affinity screening of kinase enzymes and random compound mixtures. The screening process comprises sampling and trapping equilibrium binding between candidate ligands and protein in solution, followed by removal of unbound ligands via 3 rounds of ultrafiltration and direct identification of bound ligands by mass spectrometry. Evaluation of significant peaks is facilitated by automated integration and collation of the mass spectral data and import into custom software for analysis. One Chk1-selective ligand found by using this process is presented in detail. The compound is potent in both enzymatic and Chk1-dependent cellular assays, and specific contacts in the Chk1 active site are shown by X-ray crystallography.

Acoustic sequences in non-human animals: a tutorial review and prospectus.

Animal acoustic communication often takes the form of complex sequences, made up of multiple distinct acoustic units. Apart from the well-known example of birdsong, other animals such as insects, amphibians, and mammals (including bats, rodents, primates, and cetaceans) also generate complex acoustic sequences. Occasionally, such as with birdsong, the adaptive role of these sequences seems clear (e.g. mate attraction and territorial defence). More often however, researchers have only begun to characterise - let alone understand - the significance and meaning of acoustic sequences. Hypotheses abound, but there is little agreement as to how sequences should be defined and analysed. Our review aims to outline suitable methods for testing these hypotheses, and to describe the major limitations to our current and near-future knowledge on questions of acoustic sequences. This review and prospectus is the result of a collaborative effort between 43 scientists from the fields of animal behaviour, ecology and evolution, signal processing, machine learning, quantitative linguistics, and information theory, who gathered for a 2013 workshop entitled, 'Analysing vocal sequences in animals'. Our goal is to present not just a review of the state of the art, but to propose a methodological framework that summarises what we suggest are the best practices for research in this field, across taxa and across disciplines. We also provide a tutorial-style introduction to some of the most promising algorithmic approaches for analysing sequences. We divide our review into three sections: identifying the distinct units of an acoustic sequence, describing the different ways that information can be contained within a sequence, and analysing the structure of that sequence. Each of these sections is further subdivided to address the key questions and approaches in that area. We propose a uniform, systematic, and comprehensive approach to studying sequences, with the goal of clarifying research terms used in different fields, and facilitating collaboration and comparative studies. Allowing greater interdisciplinary collaboration will facilitate the investigation of many important questions in the evolution of communication and sociality.

Combined assessment of microvascular integrity and contractile reserve improves differentiation of stunning and necrosis after acute anterior wall myocardial infarction.

OBJECTIVES: We sought to determine the relative accuracy of myocardial contrast echocardiography (MCE) and low-dose dobutamine echocardiography (LDDE) in predicting recovery of left ventricular (LV) function in patients with a recent anterior wall myocardial infarction (MI). BACKGROUND: Left ventricular dysfunction after acute MI may be secondary to myocardial stunning or necrosis. Myocardial contrast echocardiography allows real-time echocardiographic perfusion assessment from a venous injection of a fluorocarbon-based contrast agent. Although this technique is promising, it has not been compared with LDDE. METHODS: Forty-six patients underwent baseline wall motion assessment, MCE, and LDDE two days after admission, as well as follow-up echocardiography after a mean period of 53 days. RESULTS: Perfusion by MCE predicted recovery of segmental function with a sensitivity of 69%, specificity of 85%, positive predictive value of 74%, negative predictive value of 81%, and overall accuracy of 78%. Contractile reserve by LDDE predicted recovery of segmental function with a sensitivity of 50%, specificity of 88%, positive predictive value of 72%, negative predictive value of 73%, and overall accuracy of 73%. Concordant test results occurred in 74% of segments and further increased the overall accuracy to 85%. The mean wall motion score at follow-up was significantly better in perfused versus nonperfused segments (1.9 vs. 2.6, p < 0.0001) and in segments with contractile reserve, compared with segments lacking contractile reserve (1.9 vs. 2.5, p < 0.0001). CONCLUSION: Myocardial contrast echocardiography compares favorably with LDDE in predicting recovery of regional LV dysfunction after acute anterior wall MI. Concordant contractile reserve and myocardial perfusion results further enhance the diagnostic accuracy.

Diabetes mellitus is associated with a shift in the temporal risk profile of inhospital death after percutaneous coronary intervention: an analysis of 25,223 patients over 20 years.

BACKGROUND: Numerous studies have demonstrated that patients with diabetes have higher rates of restenosis, late myocardial infarction, and late death after percutaneous coronary interventions (PCI). However, it remains unclear whether patients with diabetes mellitus also have an increased hazard for early death after either elective or urgent PCI. METHODS: Patients undergoing PCI at the Mid American Heart Institute between 1980 and 1999 were identified. The main end point was inhospital death. Patients were stratified both by diabetes status and whether they underwent elective or urgent PCI. RESULTS: There were 17,341 nondiabetic patients and 4308 patients with diabetes who underwent elective PCI. There were 2946 nondiabetic patients and 628 patients with diabetes who underwent urgent PCI. Multivariate analysis demonstrated that diabetes was associated with increased inhospital mortality rate after any PCI (odds ratio 1.4, 95% CI 1.1-1.8, P =.003). The unadjusted inhospital mortality rates for the nondiabetic patients and patients with diabetes were 0.8% and 1.4%, respectively (P <.001), after elective PCI. The mortality rate was 6.9% for the nondiabetic patients and 12.7% for the patients with diabetes (P <.001) after urgent PCI. The inhospital mortality rates among diabetic patients appear to be decreasing over time among the elective cohort (elective PCI diabetes-time interaction, P =.007) but not in the urgent cohort (urgent PCI-diabetes-time interaction, P =.68). CONCLUSIONS: There has been an improvement in the inhospital survival rate among patients with diabetes in the elective PCI cohort. This improved hospital survival has yet to be realized among patients with diabetes undergoing urgent PCI.

Real-time assessment of myocardial perfusion during balloon angioplasty of the left anterior descending coronary artery.

Balloon occlusion and release during elective percutaneous coronary intervention (PCI) provides a unique opportunity to study dynamic temporal alterations in myocardial perfusion in a controlled setting. These changes in flow and volume mimic those that occur during presentation with, and successful therapy of, ST-segment elevation acute myocardial infarction (AMI). Eleven patients underwent myocardial contrast echocardiography (MCE) using a continuous infusion of Definity at baseline, during coronary occlusion, and during reactive hyperemia immediately after balloon deflation. Fifty separate flow state sequences were acquired, and off-line analysis was performed to determine myocardial contrast intensity within a region of interest in the distribution of the left anterior descending artery (LAD). A reader blinded to flow state also performed qualitative evaluation (perfusion or lack of perfusion). Quantitative analysis demonstrated significant differences in myocardial contrast intensity by flow state (p = 0.0001 for occlusion vs reperfusion). Qualitative assessment demonstrated a high rate of correct classification (92%). Real-time myocardial perfusion assessment using MCE accurately differentiates coronary occlusion and reactive hyperemia in humans by qualitative and quantitative assessment. This technique may be clinically useful in assessing the efficacy of thrombolytic therapy in ST-segment elevation AMI and in clinical trial assessment of new drugs and devices aimed at limitation of infarct size.

Assessment of the effect of ramipril therapy on direct health care costs for first and recurrent strokes in high-risk cardiovascular patients using data from the Heart Outcomes Prevention Evaluation (HOPE) study.

BACKGROUND: In the Heart Outcomes Prevention Evaluation (HOPE) Study, the angiotensin-converting enzyme (ACE) inhibitor ramipril was shown to significantly reduce the relative risk of stroke by 32% in high-risk cardiovascular patients (P < 0.001). However, the study did not examine the economic implications of these findings. OBJECTIVE: The purpose of this economic analysis was to estimate the potential economic benefits of the differences in direct health care costs attributable to the prevention of first and recurrent strokes in the HOPE Study patient population through the use of ramipril. METHODS: The epidemiologic component of the model examined the incidence of first and recurrent strokes in the HOPE Study population, assessed at annual increments, for the years 1995 through 1997. An economic decision model was constructed by the application of costs to the epidemiologic foundation. Direct costs for stroke hospitalization and follow-up were calculated based on estimates provided by Samsa et al (1999). The estimated cost of ramipril treatment was based on the average wholesale price for the corresponding year of the analysis. The Samsa index costs are given in 1991 US $; they were converted to study-year US $ using the Consumer Price Index for the corresponding year. RESULTS: The mean age of the patient population was 69 years, with >70% of patients aged >/=65 years. When ACE-inhibitor treatment costs were included in the calculation of treatment costs, the expense to avert 1 stroke was estimated at $13,766 for years 1 to 2 after randomization and $12,281 for years 2 to 3. By years 3 to 4, ramipril treatment resulted in 21 fewer strokes and produced an estimated savings of $52,861. CONCLUSION: Ramipril 10 mg/d was a cost-effective means of preventing first and recurrent ischemic strokes in the HOPE Study patient population.

Improving in-hospital mortality in the setting of an increasing risk profile among patients undergoing catheter-based reperfusion for an acute myocardial infarction without cardiogenic shock.

UNLABELLED: Prompt myocardial reperfusion is the therapeutic goal for patients presenting with acute myocardial infarction (AMI). However, there remains a paucity of clinical data from single centers solely dedicated to a catheter-based reperfusion strategy. Therefore, we sought to identify significant predictors of in-hospital mortality, to determine the changing profile of patient demographics and to identify the mortality trend over time. METHODS: Consecutive patients who underwent percutaneous coronary intervention (PCI) for an AMI between January of 1982 and December of 1999 were included in this multivariable analysis (excluding cardiogenic shock). AMI was defined as an evolving myocardial infarction within the preceding 24 hours. The primary endpoint for this analysis was in-hospital mortality. RESULTS: There were 2,745 patients identified in this study, of which 8.3% (n = 228) were non-survivors. The significant multivariable predictors of in-hospital mortality included creatinine > 1.5 mg/dl [relative risk (RR), 5.7; 95% confidence interval (CI) 4.0 8.1], ejection fraction < 40% (RR, 6.6; 95% CI, 4.3 10.0), multivessel disease (RR, 2.8; 95% CI, 1.9 4.2), female (RR, 2.3; 95% CI, 1.6 3.1) and age > 70 years (RR, 1.6; 95% CI, 1.1 2.2). The incidence of patients with these high-risk characteristics increased in recent years; thus, the unadjusted slope of the mortality trend over 20 years was not significant. However, following adjustment for the temporal shift in high-risk variables, there was a significant reduction in the adjusted in-hospital mortality rate (RR, 0.89; 95% CI 0.8 0.98; p = 0.017). Despite the changing risk profile, the short-term mortality continues to improve for patients undergoing AMI PCI.

Miniaturization of multiplexed planar recombinant antibody arrays for serum protein profiling.

BACKGROUND: Antibody-based microarrays are a developing tool for high-throughput proteomics in health and disease. However, in order to enable global proteome profiling, novel miniaturized high-density antibody array formats must be developed. RESULTS: In this proof-of-concept study, we have designed a miniaturized planar recombinant (single-chain Fragment variable). antibody array technology platform for multiplexed profiling of non-fractionated, directly labelled serum samples. The size of the individual spot features was reduced 225-times (78.5 µm(2)/spot) and the array density was increased 19-times (38,000 spots/cm(2)). These miniaturized, multiplexed arrays were produced, using a desktop nanofabrication system based on dip-pen nanolithography technology, and interfaced with a high-resolution fluorescent-based scanner. The reproducibility, sensitivity, specificity, and applicability of the set-up were demonstrated by profiling a set of well-characterized serum samples. CONCLUSION: The designed antibody array platform opens up new possibilities for large-scale, multiplex profiling of crude proteomes in a miniaturized fashion.

Direct lentiviral-cyclooxygenase 2 application to the tendon-bone interface promotes osteointegration and enhances return of the pull-out tensile strength of the tendon graft in a rat model of biceps tenodesis.

This study sought to determine if direct application of the lentiviral (LV)-cyclooxygenase 2 (COX2) vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis. The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or ßgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also significantly enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an effective therapy to promote tendon-to-bone healing after tenodesis or related surgeries.

Evaluation of the modified asthma predictive index in high-risk preschool children.

BACKGROUND: Prediction of subsequent school-age asthma during the preschool years has proven challenging. OBJECTIVE: To confirm in a post hoc analysis the predictive ability of the modified Asthma Predictive Index (mAPI) ina high-risk cohort and a theoretical unselected population. We also tested a potential mAPI modification with a 2-wheezing episode requirement (m2API) in the same populations. METHODS: Subjects (n [ 289) with a family history of allergy and/or asthma were used to predict asthma at age 6, 8, and 11 years with the use of characteristics collected during the first 3 years of life. The mAPI and the m2API were tested for predictive value. RESULTS: For the mAPI and m2API, school-age asthma prediction improved from 1 to 3 years of age. The mAPI had high predictive value after a positive test (positive likelihood ratio ranging from 4.9 to 55) for asthma development at years 6,8, and 11. Lowering the number of wheezing episodes to 2(m2API) lowered the predictive value after a positive test(positive likelihood ratio ranging from 1.91 to 13.1) without meaningfully improving the predictive value of a negative test.Posttest probabilities for a positive mAPI reached 72% and 90%in unselected and high-risk populations, respectively. CONCLUSIONS: In a high-risk cohort, a positive mAPI greatly increased future asthma probability (eg, 30% pretest probability to 90% posttest probability) and is a preferred predictive test to them 2API. With its more favorable positive posttest probability,the mAPI can aid clinical decision making in assessing future asthma risk for preschool-age children.

Discovery and characterization of non-ATP site inhibitors of the mitogen activated protein (MAP) kinases.

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

Economic impact of ramipril on hospitalization of high-risk cardiovascular patients.

OBJECTIVE: To estimate differences in direct costs attributable to avoided hospitalizations and procedures during the years of the HOPE (Heart Outcomes Prevention Evaluation) study after the cost of treatment with ramipril or alternative angiotensin-converting enzyme inhibitor therapy was taken into account. METHODS: A decision analytical model was developed to estimate the economic impact of reductions in hospitalizations and/or procedures both at annual increments and over the first 4 years of the HOPE study. The analysis compared the number of cardiovascular events per endpoint per year in the intervention and placebo group with hospitalization and procedural costs. Cost data were derived from the literature and inflated to the appropriate index year using the consumer price index. RESULTS: For approximately 9000 patients studied, the gross estimated savings in direct costs for 297 events avoided were more than $5 million over 4 years. After the cost of treatment was deducted for both groups, the net estimated savings were $871 000 over 4 years. CONCLUSIONS: The results demonstrate that the use of ramipril provides cost-effective treatment for high-risk cardiovascular patients with an ejection fraction >40%.

Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-beta-glucosyltransferase.

The BGL2 gene encodes a unique 1,3-beta-glucosyltransferase (Bgl2p) present in the cell wall of Candida albicans and other fungi. Although believed to be involved in cell wall assembly, disruption of the gene in saccharomyces cerevisiae showed no apparent phenotype. We performed sequential disruptions of the BGL2 loci in a homozygous ura3 clinical isolate of C. albicans using the URA3 blaster method, in order to investigate the role of Bgl2p in this dimorphic, pathogenic fungus. Strain CACW-1 contained disruptions of both homologues of the BGL2 gene and lacked Bgl2p, as assessed by protein extraction, SDS-PAGE and Western blot analysis, and enzyme assay; however, residual non-Bgl2p transferase activity was detected. CACW-1 was attenuated in virulence for mice when compared to an isogenic parent strain, and fewer organisms were recovered from the kidneys of infected animals. Additional phenotypic changes included: (1) a dramatic increase in the sensitivity to the chitin synthesis inhibitor nikkomycin Z when CACW-1 cells were incubated at 37 or 42 degrees C; (2) an 8.7 +/- 1.6% slower growth rate at 37 degrees C for CACW-1 when compared to its isogenic parent; and (3) aggregation of CACW-1 cells during stationary phase and/or incubation of stationary phase cells in phosphate buffer. Characterization of SDS-extracted cell walls did not reveal any significant differences in the levels of 1,3-beta- or 1,6-beta-glucan. These data reveal that loss of Bgl2p does have a phenotype in C. albicans, and indicate that (1) loss of Bgl2p function renders cells more dependent on chitin for wall integrity, and attenuates virulence (probably due to subtle changes in wall structure), and (2) that additional 1,3-beta-glucosyltransferases are present in the C. albicans BGL2 disruptant.

Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms.

Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.