The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2D(b)-restricted GP33-41 epitope of LCMV to be 1 in 2 x 10(5). Thus, in an uninfected mouse containing approximately 2-4 x 10(7) naive CD8 T cells we estimate there to be 100-200 epitope-specific cells. After LCMV infection these 100-200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of approximately 10(7) cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of approximately 5 x 10(5) cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of D(b)GP33-specific CD8 T cells from 2 x 10(2) naive cells in uninfected mice to 5 x 10(5) memory cells in immunized mice.

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (approximately 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.

BACKGROUND

Arginine vasopressin (AVP) is a key regulator of water balance, but its instability makes reliable measurement difficult and precludes routine use. We present a method for quantifying AVP release by use of copeptin, a glycopeptide comprising the C-terminal part of the AVP prohormone.

METHODS

We measured copeptin in 50-microL serum and plasma samples from healthy individuals and from critically ill patients with sepsis. Our sandwich immunoluminometric assay used 2 polyclonal antibodies to amino acids 132-164 of pre-provasopressin.

RESULTS

The assay yielded results within 3 h. The analytical detection limit was 1.7 pmol/L, and the interlaboratory CV was <20% for values >2.25 pmol/L. The assay was linear on dilution of the analyte. Ex vivo copeptin stability (<20% loss of analyte) for at least 7 days at room temperature and 14 days at 4 degrees C was shown for serum and EDTA-, heparin-, and citrate plasma. Copeptin (median, 4.2 pmol/L; range, 1-13.8 pmol/L) was detectable in 97.5% of 359 healthy individuals and was not associated with age. Median concentrations were considerably higher in men than women, increased significantly after exercise, and were influenced by fasting and water load. Copeptin was significantly (P <0.001) increased in 60 critically ill patients with sepsis (median, 79.5 pmol/L; range, 10.6-228.0 pmol/L). The correlation between copeptin and AVP for 110 samples was r = 0.78 (P <0.0001).

CONCLUSIONS

Copeptin is stable for days after blood withdrawal and can be quickly and easily measured. The copeptin assay may be a useful alternative to direct measurement of AVP concentration.

CONCLUSIONS

T-cell activation is mediated by antigen-specific signals from the TCRzeta/CD3 and CD4-CD8-p56lck complexes in combination with additional co-signals provided by coreceptors such as CD28, inducible costimulator (ICOS), cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death (PD-1), and others. CD28 and ICOS provide positive signals that promote and sustain T-cell responses, while CTLA-4 and PD-1 limit responses. The balance between stimulatory and inhibitory co-signals determines the ultimate nature of T-cell responses where response to foreign pathogen is achieved without excess inflammation and autoimmunity. In this review, we outline the current knowledge of the CD28 and CTLA-4 signaling mechanisms [involving phosphatidylinositol 3 kinase (PI3K), growth factor receptor-bound protein 2 (Grb2), Filamin A, protein kinase C theta (PKCtheta), and phosphatases] that control T-cell immunity. We also present recent findings on T-cell receptor-interacting molecule (TRIM) regulation of CTLA-4 surface expression, and a signaling pathway involving CTLA-4 activation of PI3K and protein kinase B (PKB)/AKT by which cell survival is ensured under conditions of anergy induction.

Early results of the fludarabine, cyclophosphamide, and rituximab (FCR) regimen in 224 patients showed that it was highly active as initial therapy of chronic lymphocytic leukemia. In this report, we present the final results of all 300 study patients at a median follow up of 6 years. The overall response rate was 95%, with complete remission in 72%, nodular partial remission in 10%, partial remission due to cytopenia in 7%, and partial remission due to residual disease in 6%. Two patients (< 1%) died within 3 months of starting therapy. Six-year overall and failure-free survival were 77% and 51%, respectively. Median time to progression was 80 months. Pretreatment characteristics independently associated with inferior response were age 70 years or older, beta2-microglobulin twice the upper limit of normal (2N) or more, white cell count 150 x 10(9)/L or more, abnormal chromosome 17, and lactate dehydrogenase 2N or more. No pretreatment characteristic was independently associated with decreased complete remission duration. The risk of late infection was 10% and 4% for the first and second years of remission, respectively, and less than 1.5% per year for the third year onward. In a multivariate analysis of patients receiving fludarabine-based therapy at our center, FCR therapy emerged as the strongest independent determinant of survival.

BACKGROUND

Primary care physicians report high levels of distress, which is linked to burnout, attrition, and poorer quality of care. Programs to reduce burnout before it results in impairment are rare; data on these programs are scarce.

OBJECTIVE

To determine whether an intensive educational program in mindfulness, communication, and self-awareness is associated with improvement in primary care physicians' well-being, psychological distress, burnout, and capacity for relating to patients.

METHODS

Before-and-after study of 70 primary care physicians in Rochester, New York, in a continuing medical education (CME) course in 2007-2008. The course included mindfulness meditation, self-awareness exercises, narratives about meaningful clinical experiences, appreciative interviews, didactic material, and discussion. An 8-week intensive phase (2.5 h/wk, 7-hour retreat) was followed by a 10-month maintenance phase (2.5 h/mo).

METHODS

Mindfulness (2 subscales), burnout (3 subscales), empathy (3 subscales), psychosocial orientation, personality (5 factors), and mood (6 subscales) measured at baseline and at 2, 12, and 15 months.

RESULTS

Over the course of the program and follow-up, participants demonstrated improvements in mindfulness (raw score, 45.2 to 54.1; raw score change [Delta], 8.9; 95% confidence interval [CI], 7.0 to 10.8); burnout (emotional exhaustion, 26.8 to 20.0; Delta = -6.8; 95% CI, -4.8 to -8.8; depersonalization, 8.4 to 5.9; Delta = -2.5; 95% CI, -1.4 to -3.6; and personal accomplishment, 40.2 to 42.6; Delta = 2.4; 95% CI, 1.2 to 3.6); empathy (116.6 to 121.2; Delta = 4.6; 95% CI, 2.2 to 7.0); physician belief scale (76.7 to 72.6; Delta = -4.1; 95% CI, -1.8 to -6.4); total mood disturbance (33.2 to 16.1; Delta = -17.1; 95% CI, -11 to -23.2), and personality (conscientiousness, 6.5 to 6.8; Delta = 0.3; 95% CI, 0.1 to 5 and emotional stability, 6.1 to 6.6; Delta = 0.5; 95% CI, 0.3 to 0.7). Improvements in mindfulness were correlated with improvements in total mood disturbance (r = -0.39, P < .001), perspective taking subscale of physician empathy (r = 0.31, P < .001), burnout (emotional exhaustion and personal accomplishment subscales, r = -0.32 and 0.33, respectively; P < .001), and personality factors (conscientiousness and emotional stability, r = 0.29 and 0.25, respectively; P < .001).

CONCLUSIONS

Participation in a mindful communication program was associated with short-term and sustained improvements in well-being and attitudes associated with patient-centered care. Because before-and-after designs limit inferences about intervention effects, these findings warrant randomized trials involving a variety of practicing physicians.

Pressure wave reflection in the upper limb causes amplification of the arterial pulse so that radial systolic and pulse pressures are greater than in the ascending aorta. Wave transmission properties in the upper limbs (in contrast to the descending aorta and lower limbs) change little with age, disease, and drug therapy in adult humans. Such consistency has led to use of a generalized transfer function to synthesize the ascending aortic pressure pulse from the radial pulse. Validity of this approach was tested for estimation of aortic systolic, diastolic, pulse, and mean pressures from the radial pressure waveform. Ascending aortic and radial pressure waveforms were recorded simultaneously at cardiac surgery, before initiation of cardiopulmonary bypass, with matched, fluid-filled manometer systems in 62 patients under control conditions and during nitroglycerin infusion. Aortic pressure pulse waves, generated from the radial pulse, showed agreement with the measured aortic pulse waves with respect to systolic, diastolic, pulse, and mean pressures, with mean differences <1 mm Hg. Control differences in Bland-Altman plots for mean+/-SD in mm Hg were systolic, 0.0+/-4.4; diastolic, 0.6+/-1.7; pulse, -0.7+/-4.2; and mean pressure, -0.5+/-2.0. For nitroglycerin infusion, differences respectively were systolic, -0.2+/-4.3; diastolic, 0.6+/-1.7; pulse, -0.8+/-4.1; and mean pressure, -0.4+/-1.8. Differences were within specified limits of the Association for the Advancement of Medical Instrumentation SP10 criteria. In contrast, differences between recorded radial and aortic systolic and pulse pressures were well outside the criteria (respectively, 15.7+/-8.4 and 16.3+/-8.5 for control and 14.5+/-7.3 and 15.1+/-7.3 mm Hg for nitroglycerin). Use of a generalized transfer function to synthesize radial artery pressure waveforms can provide substantially equivalent values of aortic systolic, pulse, mean, and diastolic pressures.

BACKGROUND

Postmenopausal women aged 55 years and older have 66% of incident breast tumors and experience 77% of breast cancer mortality, but other age-related health problems may affect tumor prognosis and treatment decisions.

OBJECTIVE

To document the comorbidity burden of postmenopausal breast cancer patients and evaluate its relationship with age on disease stage, treatment, and early mortality.

METHODS

Data were collected on breast cancer patients' comorbidities by retrospective hospital medical records review and merged with information on patients' tumor characteristics collected from 6 regional National Cancer Institute Surveillance, Epidemiology, and End Results cancer registries. Patients were followed up until death or for 30 months from breast cancer diagnosis.

METHODS

Population-based random sample of 1800 postmenopausal breast cancer patients diagnosed in 1992 stratified by 3 age groups: 55 to 64 years, 65 to 74 years, and 75 years and older.

METHODS

Extent of disease, therapy received, comorbidity, cause of death, and survival.

RESULTS

Seventy-three percent (1312 of 1800) of the sample was diagnosed with stage I and II breast cancer, 10% (n = 188) with stage III and IV breast cancer, and 17% (n = 300) did not have a stage assignment. Of the 1017 patients with stage I and stage II node-negative breast cancer, 95% received therapy in agreement with the National Institutes of Health consensus statement recommendation for early-stage breast cancer. Patients in older age groups were less likely to receive therapy consistent with the consensus statement (P<.001), and women aged 70 years and older were significantly less likely to receive axillary lymph node dissection as determined by logistic regression analysis (P<.01). Diabetes, renal failure, stroke, liver disease, a previous malignant tumor, and smoking were significant in predicting early mortality in a statistical model that included age and disease stage. Breast cancer was the underlying cause of death for 135 decedents (51.3%). Heart disease (n = 45, 17.1%) and previous cancers (n = 22, 8.4%) were the next major underlying causes. In the 30-month follow-up period, 263 patients (15%) died.

CONCLUSIONS

Patient care decisions occur in the context of breast cancer and other age-related conditions. Comorbidity in older patients may limit the ability to obtain prognostic information (ie, axillary lymph node dissection), tends to minimize treatment options (eg, breast-conserving therapy), and increases the risk of death from causes other than breast cancer.

BACKGROUND

Rifampin-containing regimens are able to cure staphylococcal implant-related infections based on in vitro and in vivo observations. However, this evidence has not been proven by a controlled clinical trial.

OBJECTIVE

To evaluate the clinical efficacy of a rifampin combination in staphylococcal infections associated with stable orthopedic devices.

METHODS

A randomized, placebo-controlled, double-blind trial conducted from 1992 through 1997.

METHODS

Two infectious disease services in tertiary care centers in collaboration with 5 orthopedic surgeons in Switzerland.

METHODS

A total of 33 patients with culture-proven staphylococcal infection associated with stable orthopedic implants and with a short duration of symptoms of infection (exclusion limit <1 year; actual experience 0-21 days).

METHODS

Initial debridement and 2-week intravenous course of flucloxacillin or vancomycin with rifampin or placebo, followed by either ciprofloxacin-rifampin or ciprofloxacin-placebo long-term therapy.

METHODS

Cure was defined as (1) lack of clinical signs and symptoms of infection, (2) C-reactive protein level less than 5 mg/L, and (3) absence of radiological signs of loosening or infection at the final follow-up visit at 24 months. Failure was defined as (1) persisting clinical and/or laboratory signs of infection or (2) persisting or new isolation of the initial microorganism.

RESULTS

A total of 18 patients were allocated to ciprofloxacin-rifampin and 15 patients to the ciprofloxacin-placebo combination. Twenty-four patients fully completed the trial with a follow-up of 35 and 33 months. The cure rate was 12 (100%) of 12 in the ciprofloxacin-rifampin group compared with 7 (58%) of 12 in the ciprofloxacin-placebo group (P=.02). Nine of 33 patients dropped out due to adverse events (n=6), noncompliance (n=1), or protocol violation (n=2). Seven of the 9 patients who dropped out were subsequently treated with rifampin combinations, and 5 of them were cured without removal of the device.

CONCLUSIONS

Among patients with stable implants, short duration of infection, and initial debridement, patients able to tolerate long-term (3-6 months) therapy with rifampin-ciprofloxacin experienced cure of the infection without removal of the implant.

A central mystery in the function of site-specific DNA-binding proteins is the detailed mechanism for rapid location and binding of target sites in DNA. Human oxoguanine DNA glycosylase 1 (hOgg1), for example, must search out rare 8-oxoguanine lesions to prevent transversion mutations arising from oxidative stress. Here we report high-speed imaging of single hOgg1 enzyme molecules diffusing along DNA stretched by shear flow. Salt-concentration-dependent measurements reveal that such diffusion occurs as hOgg1 slides in persistent contact with DNA. At near-physiologic pH and salt concentration, hOgg1 has a subsecond DNA-binding time and slides with a diffusion constant as high as 5 x 10(6) bp(2)/s. Such a value approaches the theoretical upper limit for one-dimensional diffusion and indicates an activation barrier for sliding of only 0.5 kcal/mol (1 kcal = 4.2 kJ). This nearly barrierless Brownian sliding indicates that DNA glycosylases locate lesion bases by a massively redundant search in which the enzyme selectively binds 8-oxoguanine under kinetic control.

OBJECTIVE

Accelerometers are recognized as a valid and objective tool to assess free-living physical activity. Despite the widespread use of accelerometers, there is no standardized way to process and summarize data from them, which limits our ability to compare results across studies. This paper a) reviews decision rules researchers have used in the past, b) compares the impact of using different decision rules on a common data set, and c) identifies issues to consider for accelerometer data reduction.

METHODS

The methods sections of studies published in 2003 and 2004 were reviewed to determine what decision rules previous researchers have used to identify wearing period, minimal wear requirement for a valid day, spurious data, number of days used to calculate the outcome variables, and extract bouts of moderate to vigorous physical activity (MVPA). For this study, four data reduction algorithms that employ different decision rules were used to analyze the same data set.

RESULTS

The review showed that among studies that reported their decision rules, much variability was observed. Overall, the analyses suggested that using different algorithms impacted several important outcome variables. The most stringent algorithm yielded significantly lower wearing time, the lowest activity counts per minute and counts per day, and fewer minutes of MVPA per day. An exploratory sensitivity analysis revealed that the most stringent inclusion criterion had an impact on sample size and wearing time, which in turn affected many outcome variables.

CONCLUSIONS

These findings suggest that the decision rules employed to process accelerometer data have a significant impact on important outcome variables. Until guidelines are developed, it will remain difficult to compare findings across studies.

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.

Rapid climate change has been implicated as a cause of evolution in poorly adapted populations. However, phenotypic plasticity provides the potential for organisms to respond rapidly and effectively to environmental change. Using a 47-year population study of the great tit (Parus major) in the United Kingdom, we show that individual adjustment of behavior in response to the environment has enabled the population to track a rapidly changing environment very closely. Individuals were markedly invariant in their response to environmental variation, suggesting that the current response may be fixed in this population. Phenotypic plasticity can thus play a central role in tracking environmental change; understanding the limits of plasticity is an important goal for future research.

BACKGROUND

Studies of people with schizophrenia assessed using magnetic resonance imaging (MRI) usually include patients with first-episode and chronic disease, yet brain abnormalities may be limited to those with chronic schizophrenia.

OBJECTIVE

To determine whether patients with a first episode of schizophrenia have characteristic brain abnormalities.

METHODS

Systematic review and meta-analysis of 66 papers comparing brain volume in patients with a first psychotic episode with volume in healthy controls.

RESULTS

A total of 52 cross-sectional studies included 1424 patients with a first psychotic episode; 16 longitudinal studies included 465 such patients. Meta-analysis suggests that whole brain and hippocampal volume are reduced (both P<0.0001) and that ventricular volume is increased (P<0.0001) in these patients relative to healthy controls.

CONCLUSIONS

Average volumetric changes are close to the limit of detection by MRI methods. It remains to be determined whether schizophrenia is a neurodegenerative process that begins at about the time of symptom onset, or whether it is better characterised as a neurodevelopmental process that produces abnormal brain volumes at an early age.

Antiangiogenic therapy has been thought to hold significant potential for the treatment of cancer. However, the efficacy of such treatments, especially in breast cancer patients, has been called into question, as recent clinical trials reveal only limited effectiveness of antiangiogenic agents in prolonging patient survival. New research using preclinical models further suggests that antiangiogenic agents actually increase invasive and metastatic properties of breast cancer cells. We demonstrate that by generating intratumoral hypoxia in human breast cancer xenografts, the antiangiogenic agents sunitinib and bevacizumab increase the population of cancer stem cells. In vitro studies revealed that hypoxia-driven stem/progenitor cell enrichment is primarily mediated by hypoxia-inducible factor 1α. We further show that the Akt/β-catenin cancer stem cell regulatory pathway is activated in breast cancer cells under hypoxic conditions in vitro and in sunitinib-treated mouse xenografts. These studies demonstrate that hypoxia-driven cancer stem cell stimulation limits the effectiveness of antiangiogenic agents, and suggest that to improve patient outcome, these agents might have to be combined with cancer stem cell-targeting drugs.

BACKGROUND

Ambrisentan is a propanoic acid-based, A-selective endothelin receptor antagonist for the once-daily treatment of pulmonary arterial hypertension.

RESULTS

Ambrisentan in Pulmonary Arterial Hypertension, Randomized, Double-Blind, Placebo-Controlled, Multicenter, Efficacy Study 1 and 2 (ARIES-1 and ARIES-2) were concurrent, double-blind, placebo-controlled studies that randomized 202 and 192 patients with pulmonary arterial hypertension, respectively, to placebo or ambrisentan (ARIES-1, 5 or 10 mg; ARIES-2, 2.5 or 5 mg) orally once daily for 12 weeks. The primary end point for each study was change in 6-minute walk distance from baseline to week 12. Clinical worsening, World Health Organization functional class, Short Form-36 Health Survey score, Borg dyspnea score, and B-type natriuretic peptide plasma concentrations also were assessed. In addition, a long-term extension study was performed. The 6-minute walk distance increased in all ambrisentan groups; mean placebo-corrected treatment effects were 31 m (P=0.008) and 51 m (P<0.001) in ARIES-1 for 5 and 10 mg ambrisentan, respectively, and 32 m (P=0.022) and 59 m (P<0.001) in ARIES-2 for 2.5 and 5 mg ambrisentan, respectively. Improvements in time to clinical worsening (ARIES-2), World Health Organization functional class (ARIES-1), Short Form-36 score (ARIES-2), Borg dyspnea score (both studies), and B-type natriuretic peptide (both studies) were observed. No patient treated with ambrisentan developed aminotransferase concentrations >3 times the upper limit of normal. In 280 patients completing 48 weeks of treatment with ambrisentan monotherapy, the improvement from baseline in 6-minute walk at 48 weeks was 39 m.

CONCLUSIONS

Ambrisentan improves exercise capacity in patients with pulmonary arterial hypertension. Improvements were observed for several secondary end points in each of the studies, although statistical significance was more variable. Ambrisentan is well tolerated and is associated with a low risk of aminotransferase abnormalities.

Association studies with candidate genes have been widely used for the study of complex diseases. However, this approach has been criticized because of non-replication of results and limits on its ability to include all possible causative genes and polymorphisms. These challenges have led to pessimism about the candidate-gene approach and about the genetic analysis of complex diseases in general. We believe that these criticisms can be usefully countered with an appeal to the principles of epidemiological investigation.

Norwalk-like viruses (NLV) are a major causative agent of nonbacterial gastroenteritis. There are still many NLV strains that are refractory to gene amplification by ordinary reverse transcription-polymerase chain reaction. This is due mainly to the genetic diversity among NLV, especially mismatches in the primer sequences, which limits this technique in clinical utility. In this study, improved primer sets based on the capsid region, to detect both genogroup I and II NLV by genogroup-specific manner, were developed. When stool specimens from gastroenteritis patients, that were positive for NLV by electron microscopy, were tested by this new primer set, all specimens were positive by RT-PCR. Primers described previously for RdRp and capsid protein were capable of amplifying the specimens by 31 and 77%, respectively. Therefore, new primer sets are extremely useful for the amplification and rapid diagnosis of nonbacterial gastroenteritis due to NLV as well as for epidemiological studies.

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

We live amid a global wave of anthropogenically driven biodiversity loss: species and population extirpations and, critically, declines in local species abundance. Particularly, human impacts on animal biodiversity are an under-recognized form of global environmental change. Among terrestrial vertebrates, 322 species have become extinct since 1500, and populations of the remaining species show 25% average decline in abundance. Invertebrate patterns are equally dire: 67% of monitored populations show 45% mean abundance decline. Such animal declines will cascade onto ecosystem functioning and human well-being. Much remains unknown about this "Anthropocene defaunation"; these knowledge gaps hinder our capacity to predict and limit defaunation impacts. Clearly, however, defaunation is both a pervasive component of the planet's sixth mass extinction and also a major driver of global ecological change.

Drug combinations are a promising strategy to overcome the compensatory mechanisms and unwanted off-target effects that limit the utility of many potential drugs. However, enthusiasm for this approach is tempered by concerns that the therapeutic synergy of a combination will be accompanied by synergistic side effects. Using large scale simulations of bacterial metabolism and 94,110 multi-dose experiments relevant to diverse diseases, we provide evidence that synergistic drug combinations are generally more specific to particular cellular contexts than are single agent activities. We highlight six combinations whose selective synergy depends on multitarget drug activity. For one anti-inflammatory example, we show how such selectivity is achieved through differential expression of the drugs' targets in cell types associated with therapeutic, but not toxic, effects and validate its therapeutic relevance in a rat model of asthma. The context specificity of synergistic combinations creates many opportunities for therapeutically relevant selectivity and enables improved control of complex biological systems.

Interferome v2.0 (http://interferome.its.monash.edu.au/interferome/) is an update of an earlier version of the Interferome DB published in the 2009 NAR database edition. Vastly improved computational infrastructure now enables more complex and faster queries, and supports more data sets from types I, II and III interferon (IFN)-treated cells, mice or humans. Quantitative, MIAME compliant data are collected, subjected to thorough, standardized, quantitative and statistical analyses and then significant changes in gene expression are uploaded. Comprehensive manual collection of metadata in v2.0 allows flexible, detailed search capacity including the parameters: range of -fold change, IFN type, concentration and time, and cell/tissue type. There is no limit to the number of genes that can be used to search the database in a single query. Secondary analysis such as gene ontology, regulatory factors, chromosomal location or tissue expression plots of IFN-regulated genes (IRGs) can be performed in Interferome v2.0, or data can be downloaded in convenient text formats compatible with common secondary analysis programs. Given the importance of IFN to innate immune responses in infectious, inflammatory diseases and cancer, this upgrade of the Interferome to version 2.0 will facilitate the identification of gene signatures of importance in the pathogenesis of these diseases.

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

OBJECTIVE

We sought to define the incidence of late clinical events and late stent thrombosis in patients treated with drug-eluting (DES) versus bare-metal stents (BMS) after the discontinuation of clopidogrel as well as their timing and outcome.

BACKGROUND

There is growing concern that delayed endothelialization after DES implantation may lead to late stent thrombosis and related myocardial infarction (MI) or death. However, event rates and outcomes after clopidogrel discontinuation versus BMS are unknown.

METHODS

A consecutive series of 746 nonselected patients with 1,133 stented lesions surviving 6 months without major events were followed for 1 year after the discontinuation of clopidogrel. Patients were assigned randomly 2:1 to DES versus BMS in BASKET (Basel Stent Kosten Effektivitäts Trial). The primary focus of this observation was cardiac death/MI.

RESULTS

Rates of 18-month cardiac death/MI were not different between DES and BMS patients. However, after the discontinuation of clopidogrel (between months 7 and 18), these events occurred in 4.9% after DES versus 1.3% after BMS implantation. Target vessel revascularization remained lower after DES, resulting in similar rates of all clinical events for this time period (DES 9.3%, BMS 7.9%). Documented late stent thrombosis and related death/target vessel MI were twice as frequent after DES versus BMS (2.6% vs. 1.3%). Thrombosis-related events occurred between 15 and 362 days after the discontinuation of clopidogrel, presenting as MI or death in 88%.

CONCLUSIONS

After the discontinuation of clopidogrel, the benefit of DES in reducing target vessel revascularization is maintained but has to be balanced against an increase in late cardiac death or nonfatal MI, possibly related to late stent thrombosis.