Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge.

Antibodies are one of the most important reagents used in biomedical and fundamental research, used to identify, and quantify proteins, contribute to knowledge of disease mechanisms, and validate drug targets. Yet many antibodies used in research do not recognize their intended target, or recognize additional molecules, compromising the integrity of research findings and leading to waste of resources, lack of reproducibility, failure of research projects, and delays in drug development. Researchers frequently use antibodies without confirming that they perform as intended in their application of interest. Here we argue that the determinants of end-user antibody choice and use are critical, and under-addressed, behavioral drivers of this problem. This interacts with the batch-to-batch variability of these biological reagents, and the paucity of available characterization data for most antibodies, making it more difficult for researchers to choose high quality reagents and perform necessary validation experiments. The open-science company YCharOS works with major antibody manufacturers and knockout cell line producers to characterize antibodies, identifying high-performing renewable antibodies for many targets in neuroscience. This shows the progress that can be made by stakeholders working together. However, their work so far applies to only a tiny fraction of available antibodies. Where characterization data exists, end-users need help to find and use it appropriately. While progress has been made in the context of technical solutions and antibody characterization, we argue that initiatives to make best practice behaviors by researchers more feasible, easy, and rewarding are needed. Global cooperation and coordination between multiple partners and stakeholders will be crucial to address the technical, policy, behavioral, and open data sharing challenges. We offer potential solutions by describing our Only Good Antibodies initiative, a community of researchers and partner organizations working toward the necessary change. We conclude with an open invitation for stakeholders, including researchers, to join our cause.

Theory of a Quantum Scanning Microscope for Cold Atoms.

We propose and analyze a scanning microscope to monitor "live" the quantum dynamics of cold atoms in a cavity QED setup. The microscope measures the atomic density with subwavelength resolution via dispersive couplings to a cavity and homodyne detection within the framework of continuous measurement theory. We analyze two modes of operation. First, for a fixed focal point the microscope records the wave packet dynamics of atoms with time resolution set by the cavity lifetime. Second, a spatial scan of the microscope acts to map out the spatial density of stationary quantum states. Remarkably, in the latter case, for a good cavity limit, the microscope becomes an effective quantum nondemolition device, such that the spatial distribution of motional eigenstates can be measured backaction free in single scans, as an emergent quantum nondemolition measurement.

Non-equilibrium 8π Josephson effect in atomic Kitaev wires.

The identification of fractionalized excitations, such as Majorana quasi-particles, would be a striking signal of the realization of exotic quantum states of matter. While the paramount demonstration of such excitations would be a probe of their non-Abelian statistics via controlled braiding operations, alternative proposals exist that may be easier to access experimentally. Here we identify a signature of Majorana quasi-particles, qualitatively different from the behaviour of a conventional superconductor, which can be detected in cold atom systems using alkaline-earth-like atoms. The system studied is a Kitaev wire interrupted by an extra site, which gives rise to super-exchange coupling between two Majorana-bound states. We show that this system hosts a tunable, non-equilibrium Josephson effect with a characteristic 8π periodicity of the Josephson current. The visibility of the 8π periodicity of the Josephson current is then studied including the effects of dephasing and particle losses.

Evidence of impairment of normal inflammatory reaction by a high-fat diet.

The murine dorsal air pouch model is a valuable tool for studying acute peripheral inflammatory reactions. We used this model to study the effect of diet on the onset of acute inflammation. Mice were fed a normal or a high-fat diet (HFD) for 5 weeks. Air pouches were raised and injected with non-stimulating (saline) or stimulating solution (saline containing lipopolysaccharides). After 4 h, leukocytes in the pouch fluid were enumerated, sorted and their viability measured. Cytokine/chemokine levels in the cell-free fluid were measured using a cytometric bead assay. Gene expression level was measured in leukocytes and in lining tissues using comparative real-time PCR. Leukocyte migration and cytokine/chemokine secretion were decreased substantially in mice fed the HFD. In contrast, leptin levels were elevated. Gene expression profiles in leukocytes recovered from the pouch and in the pouch-lining tissue (believed to have an important role in the initiation of granulocyte recruitment) were depressed. Genes encoding CC and CXC family chemokines were among the most negatively affected. These results suggest that a HFD can alter peripheral tissue activation as well as leukocyte recruitment and response, thereby affecting the development of an effective local immune response, which could have deleterious consequences.

Weak qubit measurement with a nonlinear cavity: beyond perturbation theory.

We analyze the use of a driven nonlinear cavity to make a weak continuous measurement of a dispersively coupled qubit. We calculate the backaction dephasing rate and measurement rate beyond leading-order perturbation theory using a phase-space approach which accounts for cavity noise squeezing. Surprisingly, we find that increasing the coupling strength beyond the regime describable by leading-order perturbation theory (i.e., linear response) allows one to come significantly closer to the quantum limit on the measurement efficiency. We interpret this behavior in terms of the non-Gaussian photon number fluctuations of the nonlinear cavity. Our results are relevant to recent experiments using superconducting microwave circuits to study quantum measurement.

Economic viability and critical influencing factors assessment of black water and grey water source-separation sanitation system.

The black water and grey water source-separation sanitation system aims at efficient use of energy (biogas), water and nutrients but currently lacks evidence of economic viability to be considered a credible alternative to the conventional system. This study intends to demonstrate economic viability, identify main cost contributors and assess critical influencing factors. A technico-economic model was built based on a new neighbourhood in a Canadian context. Three implementation scales of source-separation system are defined: 500, 5,000 and 50,000 inhabitants. The results show that the source-separation system is 33% to 118% more costly than the conventional system, with the larger cost differential obtained by lower source-separation system implementation scales. A sensitivity analysis demonstrates that vacuum toilet flow reduction from 1.0 to 0.25 L/flush decreases source-separation system cost between 23 and 27%. It also shows that high resource costs can be beneficial or unfavourable to the source-separation system depending on whether the vacuum toilet flow is low or normal. Therefore, the future of this configuration of the source-separation system lies mainly in vacuum toilet flow reduction or the introduction of new efficient effluent volume reduction processes (e.g. reverse osmosis).

Development of an engineering autologous palatal mucosa-like tissue for potential clinical applications.

The goal of this study was to optimize key processes in recreating functional and viable palatal mucosa-like tissue that would be easy to handle and would promote wound healing. Normal human gingival fibroblasts and epithelial cells and a clinically useful biomaterial, CollaTape, were used. Structural and ultrastructural analyses showed that the gingival fibroblasts and epithelial cells adhered to the biomaterial and proliferated. Following a 6-day culture, using 10(5) fibroblasts and 10(6) epithelial cells, a well-organized palatal mucosa-like tissue was engineered. The engineered epithelium displayed various layers, including a stratum corneum, and contained cytokeratin 16-positive cells located in the supra-basal layer. This palatal mucosa-like engineered tissue was designed to meet a variety of surgical needs. The biodegradable collagen membrane (CollaTape) contributed to the flexibility of the engineered tissue. This engineered innovative tissue may contribute to the reconstruction of oral soft-tissue defects secondary to trauma, congenital defects, and acquired diseases.

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines.

AIMS: To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity. METHODS AND RESULTS: Water samples from 40 dental units were analysed. Three different techniques for detecting Legionella spp. were compared: (i) culture approach, (ii) direct FISH and (iii) FISH with a previous R2A medium enrichment (R2A/FISH). The FISH detection was confirmed by PCR. The use of the direct FISH does not improve significantly the detection of legionellae when compared with the culture. On the contrary, when R2A/FISH was performed, sensitivity was, respectively, two- and threefold higher than that with the direct FISH and culture approach. Using R2A/FISH, 63% of water samples analysed showed a contamination by legionellae. CONCLUSIONS: Legionellae detection by direct FISH and R2A/FISH in dental unit water is possible but is more rapid and more sensitive (R2A/FISH) than the culture approach. SIGNIFICANCE AND IMPACT OF THE STUDY: R2A/FISH showed that several pathogens present in DUWL are viable but may not be culturable. Unlike PCR, R2A/FISH is designed to detect only metabolically active cells and therefore provides more pertinent information on infectious risk.

Assessment of bacterial endospore viability with fluorescent dyes.

AIM: To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. METHODS AND RESULTS: Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02). CONCLUSION: It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

Apoptosis of bronchoalveolar lavage lymphocytes in hypersensitivity pneumonitis.

The aim of this study was to look at the apoptosis of alveolar lymphocytes in hypersensitivity pneumonitis (HP). HP patients and normal unexposed controls were studied. The percentage of apoptotic lymphocytes was significantly lower in HP patients than in normal patients (37.4 +/- 3.4 versus 56.5 +/- 5.5% for Annexin V and propidium iodine detection methods and 0.4 +/- 0.1 versus 1.0 +/- 0.2% for dUTP nick end-labelling technique (TUNEL)). The proportion of bronchoalveolar lavage (BAL) lymphocytes positive for Fas antigen was significantly higher in HP patients than in normal subjects (71.7 +/- 5.4 versus 50.4 +/- 9.0%). However, no significant difference was found in the proportion of BAL lymphocytes positive for Fas ligand (FasL) between the two groups. Soluble Fas (sFas) levels in the BAL fluid of the patients and normals were 80.5 +/- 8.5 pg x mL(-1) and 23.2 +/- 3.1 pg x mL(-1), respectively. A positive correlation was found between the percentage of BAL lymphocytes and the levels of sFas for the total subjects but not within the separate study groups. The intracellular quantity of the inducible anti-apoptotic gene Bcl-xL product was significantly higher in the pulmonary lymphocytes of HP patients than in lymphocytes of the control, while no difference was found for constitutive anti-apoptotic protein (Bcl-2). In conclusion, the apoptosis of pulmonary lymphocytes is lower in hypersensitivity pneumonitis than in normal subjects. This could be explained, at least in part, by an increase of soluble Fas, the anti-apoptic gene, and Bcl-xL.

Catheter-assisted totally thoracoscopic coronary artery bypass grafting: a feasibility study.

BACKGROUND: The purpose of this study is to examine the feasibility of performing totally thoracoscopic internal mammary-to-coronary artery bypass grafting with the assistance of radiologically guided catheter intervention. METHODS: Fourteen dogs were subjected to mobilization of the internal mammary artery and anastomosis of it to the left anterior descending coronary artery over an angiographic catheter inserted into the internal mammary artery under fluoroscopy. The anastomosis was completed over the catheter using sutures and the application of fibrin glue. Eight animals underwent the anastomosis after their sacrifice. The other 6 animals were put on closed chest cardiopulmonary bypass and had their anastomosis done after intraaortic balloon occlusion and cardioplegic arrest of the heart. All animals had an angiographic and pathologic examination at the completion of the anastomosis. RESULTS: Anastomosis was completed in all dogs. Three anastomoses leaked and two were noted to be stenosed at completion of the anastomosis. One leak was sealed by application of fibrin glue. Both stenotic anastomoses were caused by suturing of the back wall when a short angiographic catheter could not be positioned across the anastomosis. CONCLUSIONS: Minimally invasive totally thoracoscopic mammary-to-coronary artery bypass grafting with catheter assistance is feasible. Technical improvement and appropriate instrumentation are required to minimize anastomotic failure.

Electrocardiographic and hemodynamic changes and their association with myocardial infarction during coronary artery bypass surgery. A multicenter study. Multicenter Study of Perioperative Ischemia (McSPI) Research Group.

BACKGROUND: Electrocardiographic (ECG) changes during coronary artery bypass graft surgery have not been described in detail in a large multicenter population. The authors describe these ECG changes and evaluate them, along with demographic and clinical characteristics and intraoperative hemodynamic alterations, as predictors of myocardial infarction (MI) as defined by two sets of criteria. METHODS: Data from 566 patients at 20 clinical sites, collected as part of a clinical trial to evaluate the efficacy of acadesine for reducing MI, were analyzed at core laboratories. Perioperative ECG changes were identified using continuous three-lead Holter ECG. Systolic blood pressure, diastolic blood pressure, and heart rate were recorded each minute during operation. The occurrence of MI by Q wave or myocardial fraction of creatine kinase (CK-MB) or autopsy criteria, and by (Q wave and CK-MB) or autopsy criteria was determined. RESULTS: During perioperative Holter monitoring, episodes of ST segment deviation, major cardiac conduction changes > or = 30 min, or use of ventricular pacing > or = 30 min occurred in 58% patients, primarily in the first 8 h after release of aortic occlusion. Of the 25% patients who met the Q wave or CK-MB or autopsy criteria for MI, 19% had increased CK-MB as well as ECG changes. (Q wave and CK-MB) or autopsy criteria for MI were met by 4% of patients. The CK-MB concentration generally peaked by 16 h after release of aortic occlusion. In patients with (n = 187) and without a perioperative episode of ST segment deviation, the incidence of MI was 36% and 19%, respectively (P < 0.01), by Q wave or CK-MB or autopsy criteria, and 6% and 3%, respectively (P = 0.055), by (Q wave and CK-MB) or autopsy criteria. Multiple logistic regression analysis showed that intraoperative ST segment deviation, intraventricular conduction defect, left bundle branch block, duration of hypotension (systolic blood pressure < 90 mmHg) after cardiopulmonary bypass, and duration of cardiopulmonary bypass are independent predictors of Q wave or CK-MB or autopsy MI. The independent predictors of (Q wave and CK-MB) or autopsy MI are intraoperative ST segment deviation and duration of aortic occlusion. CONCLUSIONS: Major ECG changes occurred in 58% of patients during coronary artery bypass graft surgery, primarily within 8 h after release of aortic occlusion. Multicenter data collection revealed a substantial variation in the incidence of MI and an overall incidence of up to 25%, with most MI occurring within 16 h after release of aortic occlusion. Intraoperative monitoring of ECG and hemodynamics has incremental value for predicting MI.