Phenotype-Based Threat Assessment.

Bacterial pathogen identification, which is critical for human health, has historically relied on culturing organisms from clinical specimens. More recently, the application of machine learning (ML) to whole-genome sequences (WGSs) has facilitated pathogen identification. However, relying solely on genetic information to identify emerging or new pathogens is fundamentally constrained, especially if novel virulence factors exist. In addition, even WGSs with ML pipelines are unable to discern phenotypes associated with cryptic genetic loci linked to virulence. Here, we set out to determine if ML using phenotypic hallmarks of pathogenesis could assess potential pathogenic threat without using any sequence-based analysis. This approach successfully classified potential pathogenetic threat associated with previously machine-observed and unobserved bacteria with 99% and 85% accuracy, respectively. This work establishes a phenotype-based pipeline for potential pathogenic threat assessment, which we term PathEngine, and offers strategies for the identification of bacterial pathogens.

Prediction of whole-cell transcriptional response with machine learning.

MOTIVATION: Applications in synthetic and systems biology can benefit from measuring whole-cell response to biochemical perturbations. Execution of experiments to cover all possible combinations of perturbations is infeasible. In this paper, we present the host response model (HRM), a machine learning approach that maps response of single perturbations to transcriptional response of the combination of perturbations. RESULTS: The HRM combines high-throughput sequencing with machine learning to infer links between experimental context, prior knowledge of cell regulatory networks, and RNASeq data to predict a gene's dysregulation. We find that the HRM can predict the directionality of dysregulation to a combination of inducers with an accuracy of >90% using data from single inducers. We further find that the use of prior, known cell regulatory networks doubles the predictive performance of the HRM (an R2 from 0.3 to 0.65). The model was validated in two organisms, Escherichia coli and Bacillus subtilis, using new experiments conducted after training. Finally, while the HRM is trained with gene expression data, the direct prediction of differential expression makes it possible to also conduct enrichment analyses using its predictions. We show that the HRM can accurately classify >95% of the pathway regulations. The HRM reduces the number of RNASeq experiments needed as responses can be tested in silico prior to the experiment. AVAILABILITY AND IMPLEMENTATION: The HRM software and tutorial are available at https://github.com/sd2e/CDM and the configurable differential expression analysis tools and tutorials are available at https://github.com/SD2E/omics_tools. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Monitoring cell-mediated immunity during immunosuppression reduction in heart transplant recipients with severe systemic infections.

BACKGROUND: Treatment for severe systemic infections in heart transplantation is reduction in immunosuppression while treating the infection. An assay that measures adenosine triphosphate production in activated lymphocytes (ImmuKnow® ) objectively monitors cellular immunity of transplant recipients. In this study, we used ImmuKnow® to adjust immunosuppression in heart transplant recipients with severe systemic infections. METHODS: Heart transplant recipients were followed with ImmuKnow® at the time of biopsy and diagnosis of systemic infection. Patients who developed an infection were monitored by ImmuKnow® assay with adjustments in immunosuppression based upon the results of the assay. Maintenance immunosuppression was reinstituted when the ImmuKnow® increased to >225 ng/mL of ATP. RESULTS: Two or more ImmuKnow® assays were performed in 80 patients. Thirteen patients developed severe systemic infections. ImmuKnow® mean value at the time of diagnosis of infection was 109 ± 49.2 ng/mL. Reduction in immunosuppression and treatment of infection resulted in normalization of ImmuKnow® level, resolution of infection, and no episodes of rebound rejection. CONCLUSION: Heart transplant recipients with severe systemic infections presented with a decreased ImmuKnow® , suggesting over immunosuppression. ImmuKnow® can be used as an objective measurement in withdrawing immunosuppression in heart transplant recipients with severe systemic infections.

Translucent poly(vinyl alcohol) cryogel dosimeters for simultaneous dose buildup and monitoring during chest wall radiation therapy.

Chest wall radiation therapy treatment delivery was monitored using a 5 mm thick radiochromic poly(vinyl alcohol) cryogel that also provided buildup material. The cryogels were used to detect positioning errors and measure the impact of shifts for a chest wall treatment that was delivered to a RANDO phantom. The phantom was shifted by ± 2, ± 3, and ± 5 mm from the planned position in the anterior/posterior (A/P) direction; these shifts represent setup errors and the uncertainty associated with lung filling during breath-hold. The two-dimensional absolute dose distributions measured in the cryogel at the planned position were compared with the distributions at all shifts from this position using gamma analysis (3%/3 mm, 10% threshold). For shifts of ± 2, ± 3, and ± 5 mm the passing rates ranged from 94.3% to 95.6%, 74.0% to 78.8%, and 17.5% to 22.5%, respectively. These results are consistent with the same gamma analysis performed on dose planes calculated in the middle of the cryogel and on the phantom surface using our treatment plan-ning system, which ranged from 94.3% to 95.0%, 76.8% to 77.9%, and 23.5% to 24.3%, respectively. The Pinnacle dose planes were then scaled empirically and compared to the cryogel measurements. Using the same gamma metric, the pass rates ranged from 97.0% to 98.4%. The results of this study suggest that cryogels may be used as both a buildup material and to evaluate errors in chest wall treat-ment positioning during deep-inspiration breath-hold delivery. The cryogels are sensitive to A/P chest wall shifts of less than 3 mm, which potentially allows for the detection of clinically relevant errors.

Effect of 1O2 quencher depletion on the efficiency of photodynamic therapy.

Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4) fluorescence and photodynamic oxygen consumption were monitored during AlPcS4-photodynamic therapy (PDT) of Mat LyLu cells in suspension. These measurements were used to calculate the PDT efficiency, which is defined as the oxygen consumption rate divided by the sensitizer concentration. As a function of the intracellular oxygen concentration consumed by PDT, the normalized PDT efficiency fell off more quickly at lower photosensitizer concentrations. The changes in PDT efficiency were compared to models of PDT in which the photosensitizer (PS) and singlet oxygen quencher (A) were either free to diffuse or were fixed. The model in which PS and A are free to diffuse did not agree with the experimental data because this model predicts that the reduction in PDT efficiency is independent of [PS]. A Monte Carlo model was written to simulate PDT when both PS and A are stationary. This model was found to describe the experimental data when the initial intracellular [A] = 90 mM and when the initial and final (i.e. after all A has been depleted) singlet oxygen lifetimes were 0.4 and 1.2 µs respectively.

Peer workers' role in smoking-cessation groups for people with mental illness.

OBJECTIVES: The objective of this article is to evaluate the impact of peer workers' involvement as co-leaders in smoking-cessation programmes provided within mental health services. METHOD: Group smoking-cessation programmes were provided for people living with mental illness. Peer workers were involved in the development and delivery of these programmes. Group participants and mental health workers were asked to respond to a questionnaire about their experience of the peer workers. The questionnaire included both Likert scales and qualitative responses. RESULTS: Thirty-three mental health workers and 108 group participants completed the questionnaire. The majority of participants believed that the peer workers increased their confidence, helped them to learn about smoking cessation and promoted well-being. Mental health workers were also positive about the role of peer workers in the groups. CONCLUSIONS: This study supports the role of peer workers providing support and guidance within smoking-cessation programmes for people with mental illness. The results suggest that peer workers make a substantial contribution and that greater peer worker involvement in such programmes is likely to improve their acceptability and efficacy.

Neural network based integration of assays to assess pathogenic potential.

Limited data significantly hinders our capability of biothreat assessment of novel bacterial strains. Integration of data from additional sources that can provide context about the strain can address this challenge. Datasets from different sources, however, are generated with a specific objective and which makes integration challenging. Here, we developed a deep learning-based approach called the neural network embedding model (NNEM) that integrates data from conventional assays designed to classify species with new assays that interrogate hallmarks of pathogenicity for biothreat assessment. We used a dataset of metabolic characteristics from a de-identified set of known bacterial strains that the Special Bacteriology Reference Laboratory (SBRL) of the Centers for Disease Control and Prevention (CDC) has curated for use in species identification. The NNEM transformed results from SBRL assays into vectors to supplement unrelated pathogenicity assays from de-identified microbes. The enrichment resulted in a significant improvement in accuracy of 9% for biothreat. Importantly, the dataset used in our analysis is large, but noisy. Therefore, the performance of our system is expected to improve as additional types of pathogenicity assays are developed and deployed. The proposed NNEM strategy thus provides a generalizable framework for enrichment of datasets with previously collected assays indicative of species.

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.

Computational tools addressing various components of design-build-test-learn (DBTL) loops for the construction of synthetic genetic networks exist but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called Design Assemble Round Trip (DART). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions. Graphical Abstract.

Intent Parser: A Tool for Codification and Sharing of Experimental Design.

Communicating information about experimental design among a team of collaborators is challenging because different people tend to describe experiments in different ways and with different levels of detail. Sometimes, humans can interpret missing information by making assumptions and drawing inferences from information already provided. Doing so, however, is error-prone and typically requires a high level of interpersonal communication. In this paper, we present a tool that addresses this challenge by providing a simple interface for incremental formal codification of experiment designs. Users interact with a Google Docs word-processing interface with structured tables, backed by assisted linking to machine-readable definitions in a data repository (SynBioHub) and specification of available protocols and requests for execution in the Open Protocol Interface Language (OPIL). The result is an easy-to-use tool for generating machine-readable descriptions of experiment designs with which users in the DARPA SD2 program have collected data from 80 208 samples using a variety of protocols and instruments over the course of 181 experiment runs.

Round Trip: An Automated Pipeline for Experimental Design, Execution, and Analysis.

Synthetic biology is a complex discipline that involves creating detailed, purpose-built designs from genetic parts. This process is often phrased as a Design-Build-Test-Learn loop, where iterative design improvements can be made, implemented, measured, and analyzed. Automation can potentially improve both the end-to-end duration of the process and the utility of data produced by the process. One of the most important considerations for the development of effective automation and quality data is a rigorous description of implicit knowledge encoded as a formal knowledge representation. The development of knowledge representation for the process poses a number of challenges, including developing effective human-machine interfaces, protecting against and repairing user error, providing flexibility for terminological mismatches, and supporting extensibility to new experimental types. We address these challenges with the DARPA SD2 Round Trip software architecture. The Round Trip is an open architecture that automates many of the key steps in the Test and Learn phases of a Design-Build-Test-Learn loop for high-throughput laboratory science. The primary contribution of the Round Trip is to assist with and otherwise automate metadata creation, curation, standardization, and linkage with experimental data. The Round Trip's focus on metadata supports fast, automated, and replicable analysis of experiments as well as experimental situational awareness and experimental interpretability. We highlight the major software components and data representations that enable the Round Trip to speed up the design and analysis of experiments by 2 orders of magnitude over prior ad hoc methods. These contributions support a number of experimental protocols and experimental types, demonstrating the Round Trip's breadth and extensibility. We describe both an illustrative use case using the Round Trip for an on-the-loop experimental campaign and overall contributions to reducing experimental analysis time and increasing data product volume in the SD2 program.

Highly-automated, high-throughput replication of yeast-based logic circuit design assessments.

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of Saccharomyces cerevisiae by Gander et al. Our experimental campaign used a novel high-throughput experimentation framework developed under Defense Advanced Research Projects Agency's Synergistic Discovery and Design program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report and make some remarks about lessons learned. Graphical Abstract.

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist.

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.

Immuno-Electrophysiological Mechanisms of Functional Electrical Connections Between Recipient and Donor Heart in Patients With Orthotopic Heart Transplantation Presenting With Atrial Arrhythmias.

[Figure: see text].

Framing male circumcision to promote its adoption in different settings.

The effectiveness of male circumcision in preventing transmission of HIV from females to males has been established. Those who are now advocating its widespread use face many challenges in convincing policy-makers and the public of circumcision's value. We suggest that frames are a useful lens for communicating public health messages that may help promote adoption of circumcision. Frames relate to how individuals and societies perceive and understand the world. Existing frames are often hard to shift, and should be borne in mind by advocates and program implementers as they attempt to promote male circumcision by invoking new frames. Frames differ across and within societies, and advocates must find ways of delivering resonant messages that take into account prior perceptions and use the most appropriate means of communicating the benefits and value of male circumcision to different audiences.

Male circumcision and HIV prevention: looking to the future.

Now that male circumcision has been shown to have a protective effect for men against HIV infection when engaging in vaginal intercourse with HIV-infected women, the research focus needs to shift towards the operational studies that can pave the way for effective implementation of circumcision programs. Behavioral research is needed to find out how people perceive the procedure and the barriers to and facilitators of uptake. It should also assess the risk of an increase in unsafe sex after circumcision. Social research must examine cultural perceptions of the practice, in Africa and beyond, including how likely uncircumcised communities are to access surgery and what messages are needed to persuade them. Advocates of male circumcision would benefit from research on how to influence health policy-makers, how best to communicate the benefits to the public, and how to design effective delivery models.

Site-specific differences in latency intervals during biventricular pacing: impact on paced QRS morphology and echo-optimized V-V interval.

OBJECTIVE: To investigate differences in latency intervals during right ventricular (RV) pacing and left ventricular (LV) pacing from the (postero-)lateral cardiac vein in cardiac resynchronization therapy (CRT) patients and their relationship to echo-optimized interventricular (V-V) intervals and paced QRS morphology. METHODS: We recorded digital 12-lead electrocardiograms in 40 CRT patients during RV, LV, and biventricular pacing at three output settings. Stimulus-to-earliest QRS deflection (latency) intervals were measured in all leads. Echocardiographic atrioventricular (AV) and V-V optimization was performed using aortic velocity time integrals. RESULTS: Latency intervals were longer during LV (34 ± 17, 29 ± 15, 28 ± 15 ms) versus RV apical pacing (17 ± 8, 15 ± 8, 13 ± 7 ms) for threshold, threshold ×3, and maximal output, respectively (P < 0.001), and shortened with increased stimulus strength (P < 0.05). The echo-optimized V-V interval was 58 ± 31 ms in five of 40 (12%) patients with LV latency ≥ 40 ms compared to 29 ± 20 ms in 35 patients with LV latency < 40 ms (P < 0.01). During simultaneous biventricular pacing, four of five (80%) patients with LV latency ≥ 40 ms exhibited a left bundle branch block (LBBB) pattern in lead V(1) compared to three of 35 (9%) patients with LV latency < 40 ms (P < 0.01). After optimization, all five patients with LV latency ≥ 40 ms registered a dominant R wave in lead V(1) . CONCLUSIONS: LV pacing from the lateral cardiac vein is associated with longer latency intervals than endocardial RV pacing. LV latency causes delayed LV activation and requires V-V interval adjustment to improve hemodynamic response to CRT. Patients with LV latency ≥ 40 ms most often display an LBBB pattern in lead V(1) during simultaneous biventricular pacing, but a right bundle branch block after V-V interval optimization.

Simple photodynamic therapy dose models fail to predict the survival of MLL cells after HPPH-PDT in vitro.

Fluorescence photobleaching, photodynamic therapy (PDT) oxygen consumption and clonogenic cell survival were investigated during 2-(1-hexyloxethyl)-2-devinyl pyropheophoribde-a (HPPH) PDT of MAT-LyLu cells in vitro. Cells were incubated with HPPH concentrations of 0.24, 1.2, 3.6 or 12 microm for 4 h and then treated with 650 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment and photobleaching was quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony forming assay. Intracellular fluorescence lifetime measurements were also performed at each incubation concentration. The photobleaching kinetics did not follow first- or second-order kinetics and the fluorescence lifetime was similar for all intracellular concentrations. As the intracellular concentration of drug was increased, the amount of singlet oxygen and the absorbed quanta per cell required to achieve the same cell kill increased. Singlet oxygen dose was calculated using one- and two-compartment models of HPPH intracellular distribution. It was found that a two-compartment model, in which a PDT-sensitive binding site saturates at low concentrations, accounts for the observed photobleaching, oxygen consumption and cell survival.

Rapid access towards follow-up NOP receptor agonists using a knowledge based approach.

A knowledge based approach has been adopted to identify novel NOP receptor agonists with simplified hydrophobes. Substitution of the benzimidazol-2-one piperidine motif with a range of hydrophobic groups and pharmacophore guided bio-isosteric replacement of the benzimidazol-2-one moiety was explored. Compound 51 was found to be a high affinity, potent NOP receptor agonist with reduced affinity for the hERG channel.

Outcomes of Induction Therapy with Rabbit Anti-Thymocyte Globulin in Heart Transplant Recipients: A Single Center Retrospective Cohort Study.

BACKGROUND Induction immunosuppression is used in transplantation to prevent early acute rejection. The survival benefit of rabbit anti-thymocyte globulin (rATG) induction has not been established yet. We sought to determine the role of rATG in preventing rejection and improving overall survival. MATERIAL AND METHODS A retrospective cohort study was conducted from 2005 to 2009 and data of consecutive 268 heart transplant recipients were reviewed. RESULTS The data of 144 patients who received induction with rATG were compared to 124 patients who did not. Although overall survival was not different between the 2 groups (P=0.12), there was a significant difference in restricted mean survival time (RMST) at 5 years (RMST=4.8 months; 95% CI: 1.0-8.6, P=0.01) and 10 years (RMST=10.4 months; 95% CI: 1.6-19.3, P=0.02) in favor of the non-induced patients. No difference was observed between induced and non-induced patients who developed de novo donor specific antibodies. There was a significant difference in median days to first rejection in favor of the induced group (P<0.001). CONCLUSIONS Induction with rATG adds no survival benefit in heart transplant recipients. Patients who did not receive induction therapy had higher life expectancy at 5 years and 10 years. Although there was significant delay in the first rejection episode in favor of the rATG induced group, no difference was observed in donor specific antibodies. This study indicates a need for separate analysis of peri-transplantation co-morbidities and mainly the incidence of acute kidney injury, which could affect long-term survival.

Business and AIDS: sectoral challenges and opportunities.

The Business and AIDS think tank held in Durban, South Africa, in June 2006, included a discussion of the policies with which different types of employer could address HIV/AIDS in southern Africa. Breakout groups discussed the role of large and small private sector firms, the public sector, and parastatal organizations. They made recommendations for policies, programmes and future research for each sector.