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This document presents hydrogen-deuterium exchange mass spectrometry (HDX-MS) data from measurements of three purified IgG1 glycoform samples, predominantly G0F, G2F, and SAF, in isolation and in complexation with the high-affinity receptor, FcγRIa (CD64). The IgG1 antibody used in this study, aIL8hFc, is a murine-human chimeric IgG1, which inhibits IL-8 binding to human neutrophils.

We present the CamilleX framework for the Rodin platform in this paper. The framework provides a textual representation and persistence for the Event-B modelling constructs. It supports direct extensions to the Event-B syntax such as machine inclusion and record structures, as well as indirect extensions provided by other plugins such as UML-B diagrams. We discusses CamilleX’s design and in particular, its extension mechanisms and examples of their use.

The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed-batch cultures. The activity level of Na+ /K+ ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a markerless indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary (CHO) cells during nutrient deprivation and reintroduction. Employing single cell dielectrophoresis, the change in cytoplasm conductivity was measured over a 48-hr period. The conditions under which the cytoplasm conductivity would recover to a normal level after nutrient reintroduction was determined. In addition, numerical simulations of cell ion flux, for different levels of Na+ /K+ ATPase pump inhibition, were used to predict the minimum conductivity expected for nutrient-deprived CHO cells. This predicted value is close to the minimum observed experimental cytoplasm conductivity for CHO cells that maintain the ability to restore the cytoplasm conductivity to the normal viable levels when nutrients are reintroduced. The recovery of starved cells was verified by reintroducing them to nutrient for 36 hr and measuring their proliferation using trypan blue exclusion assay. We conclude that cytoplasm conductivity can be used as a marker to indicate whether cells are in a recoverable state, such that the reintroduction of nutrients results in cells returning to a normal healthy state.

The dielectric properties of cells are directly related to their morphological and physiological properties and can be used to monitor their status when exposed to stress conditions. In this work, dual-frequency dielectrophoresis (DEP) cytometry was employed to measure changes in the membrane capacitance and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells during the progression of starvation-induced apoptosis. Our dual-frequency DEP cytometer enables simultaneous measurement of multiple dielectric properties of single cells and identification of their state (viable or apoptotic) within a heterogeneous sample. We employed one frequency to determine each cell's viability state and the other frequency to characterize the change in membrane capacitance or cytoplasm conductivity. Cells were starved by incubation in a medium lacking glucose and glutamine and monitored every 12 h over a 64 h period. Our results showed a subpopulation of early apoptotic cells emerged after 40 h in the starvation medium, which rapidly increased during the next 12 h. After 52 h, a complete transition from viable to apoptotic state was observed. Analyzing the subpopulation of viable cells over the first 52 h showed that the membrane capacitance gradually declined from an initial value of 2.0 to 1.2 μF/cm2, and was 0.9 μF/cm2 for apoptotic cells. The cytoplasm conductivity of viable cells initially remained constant and then declined from 0.40 to 0.27 S/m after 40 h, coinciding with onset of apoptotic processes. A dramatic decrease in cytoplasm conductivity from 0.27 to 0.07 S/m was observed after 52 h, corresponding to apoptotic cells. As membrane capacitance is related to membrane morphology and cytoplasm conductivity is related to intracellular ion concentrations, the results indicate that during controlled starvation the cell membrane smooths gradually whereas intracellular ion concentrations are initially maintained near homeostatic levels until a later dramatic decline occurs.

Dielectrophoresis (DEP) cytometry was used to investigate changes in the membrane capacitance, membrane conductance, and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells subjected to thermal stress. CHO cells were exposed to temperatures ranging from 37 °C to 50 °C for 15 min and measured immediately after exposure. Heat-treated cells were measured at frequencies over the $\beta $ -dispersion region to obtain the dielectric parameters of a multishell cell model. A microwave DEP cytometer measuring DEP-induced translation of many individual heat-treated cells at selected frequencies was used to determine the Clausius–Mossotti factor and subsequently cell dielectric parameters. Results reveal that temperature-induced stress is accompanied by decrease in cell membrane capacitance from 1.75 to $0.71~\mu \text{F}$ /cm2, suggesting alteration in morphological features of the cell membrane surface. Results also show an insignificant decline in cytoplasm conductivity for treatment up to 46 °C. A significant decline in cytoplasm conductivity, from an initial value of 0.34–0.22 S/m was observed for 50 °C. The effect of suspending cells in low-conductivity DEP media was also investigated.

Event-B, a refinement-based formal modelling language, has traditionally focused on safety, but now increasingly finds a new role in developing secure systems. In this paper we take a fresh look at security and focus on what security means for the system rather than looking at detailed protocols. We use Event-B for proving security from an abstract view and refining it towards design details, focusing on the refinement of the availability property of the system. We define a general approach to guarantee the availability of events by ensuring the non-strengthening of their guards, taking into consideration their parameter types.We illustrate our approach using a smart ballot system, an integral part of modern voting systems.

Formal methods use abstraction and rigorously verified refinement to manage the design of complex systems, ensuring that they satisfy important invariant properties. However, formal verification is not sufficient: models must also be tested to ensure that they behave according to the informal requirements and validated by domain experts who may not be expert in formal modelling. This can be satisfied by scenarios that complement the requirements specification. The model can be animated to check whether the scenario is feasible in the model and that the model reaches the states expected in the scenario. However, there are two problems with this approach. 1) The natural language used to describe the scenarios is often verbose, ambiguous and therefore difficult to understand; especially if the modeller is not a domain expert. 2) Provided scenarios are typically at the most concrete level corresponding to the full requirements and cannot be used until all the refinements have been completed in the model. We show by example how a precise and concise domain specific language can be used for writing these abstract scenarios in a style that can be easily understood by the domain expert (for validation purposes) as well as the modeller (for behavioural verification) and can be used as the persistence for automated tool support. We propose two alternative approaches to using scenarios during formal modelling: A method of refining scenarios before the model is refined so that the scenarios guide the modelling, and a method of abstracting scenarios from provided concrete ones so that they can be used to test early refinements of the model. We illustrate the two approaches on the ‘Tokeneer’ secure enclave example and the ERTMS/ETCS Hybrid Level 3 specification for railway controls. We base our approach on the Cucumber framework for scenarios and the Event-B modelling language and tool set. We have developed a new ‘Scenario Checker’ plugin to manage the animation of scenarios. 1

In this document, we propose a universal definition of heart failure (HF) as a clinical syndrome with symptoms and/or signs caused by a structural and/or functional cardiac abnormality and corroborated by elevated natriuretic peptide levels and/or objective evidence of pulmonary or systemic congestion. We also propose revised stages of HF as: At risk for HF (Stage A), Pre-HF (Stage B), Symptomatic HF (Stage C) and Advanced HF (Stage D). Finally, we propose a new and revised classification of HF according to left ventricular ejection fraction (LVEF). This includes HF with reduced ejection fraction (HFrEF): symptomatic HF with LVEF = 50%; and HF with improved ejection fraction (HFimpEF): symptomatic HF with a baseline LVEF = 10 point increase from baseline LVEF, and a second measurement of LVEF > 40%.

State-based formal specifications benefit from data structuring mechanisms, which collate associated properties and efficiently declare complex types. For example, ‘record’ data structures, similar to those used in programming languages, can be built into the concrete syntax of a language as an enhancement over flat data relationships. While this is relatively simple to achieve for a single-level specification, it becomes significantly more involved when the specification language allows for progressive refinement of the data supporting the specification. Individual fields may be added to create sub-records within a refinement and replaced to create refined records during a refinement step. The impact on the ability to verify invariant and refinement proof obligations must be considered. Here we describe a record structuring syntax that includes notions of extension and inheritance that can be used in a refinement-based formal method. We illustrate the approach using extensions to the Event-B formal method.

Event-B is a state-based formal method for system development. The Event-B mathematical language does not support a syntax for the direct definition of structured types such as records. This paper proposes extending the Event-B language with direct record definitions. A key feature is the ability to extend records with new fields in refinement steps. The XEvent-B tool, which supports a textual representation of Event-B models, is extended to provide support for direct record definition and automatic transformation of record structures into standard Event-B elements. We demonstrate this work by modelling of the Tokeneer case study.

As the physical world evolves with time, safety-critical systems are usually used with time-dependent functionality. The design and implementation of real-time systems are challenging due to the complicated functional and timing requirements. Event - B formalization offers a stepwise development approach for specifying and verifying systems with mathematical techniques and tools. In this paper, we propose four realtime specification patterns, namely time response pattern, abort pattern, intermediate pattern and periodic pattern, to facilitate the specification of real-time properties in Event-B models. The proposed patterns are used in a dual-chamber pacemaker case study to specify and verify the timing cycles based on the requirements. The model is proved using the Rodin tool.

The control of glycosylation profiles is essential to the consistent manufacture of therapeutic monoclonal antibodies that may be produced from a variety of cell lines including CHO and NS0. Of particular concern is the potential for generating non-human epitopes such as N-glycolylneuraminic acid (Neu5Gc) and Galα1-3 Gal that may be immunogenic. We have looked at the effects of a commonly used media supplements of manganese, galactose and uridine (MGU) on Mab production from CHO and NS0 cells in enhancing galactosylation and sialylation as well as the generation of these non-human glycan epitopes. In the absence of the MGU supplement, the humanized IgG1 antibody (Hu1D10) produced from NS0 cells showed a low level of mono- and di-sialylated structures (SI:0.09) of which 75 % of sialic acid was Neu5Gc. The chimeric human-llama Mab (EG2-hFc) produced from CHO cells showed an equally low level of sialylation (SI: 0.12) but the Neu5Gc content of sialic acid was negligible (

Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. Differences between cultures appeared in the decline phase which was followed up to 7 days beyond the start of the cultures. There was a marginal but significant overall increase (x1.1) in the integral viable cell density (IVCD) in the presence of hydrolysate but a more substantial increase in the cell-specific mAb (qMab) productivity (x1.5). There were individual differences between hydrolysates in terms of enhancement of mAb productivity, the highest being a 166% increase of mAb titre (to 117 mg/L) in batch cultures of CHO-EG2 supplemented with UPcotton hydrolysate. The effect of one of the most active hydrolysates (HP7504) on antibody glycosylation was investigated. This showed no change in the predominant seven glycans produced but a significant increase in the galactosylation and sialylation of some but not all the antibodies. Overall, the animal hydrolysate, Primatone and two cotton-derived hydrolysates provided the most substantial benefit for enhanced productivity. The cotton-based hydrolysates can be viewed as valuable supplements for animal-derived component-free (ADCF) media and as a source for the investigation of chemically defined bioactive components. • Protein hydrolysates enhanced both IVCD & qMab; the effect on qMab being consistently greater. • Cotton-based hydrolysates showed high bioactivity and potential for use in serum-free media. • Enhanced galactosylation and sialylation was shown for some of the Mabs tested.

Previous reports present different models for the stabilization of the Fc-FcγRI immune complex. Although accord exists on the importance of L235 in IgG1 and some hydrophobic contacts for complex stabilization, discord exists regarding the existence of stabilizing glycoprotein contacts between glycans of IgG1 and a conserved FG-loop (

The Theory plug-in for the Rodin Platform enables modellers to extend the mathematical modelling notation for Event-B, with accompanying support for reasoning about the extended language. We consider in this chapter using Rodin theories to capture domain-specific Abstract Data Types (ADTs) and build dynamic systems using the developed structures. In particular, we propose the notion of theory instantiation to incorporate more concrete representation of the ADTs. At the same time, the dynamic systems are refined further with respect to the changes of the underlying ADTs. We illustrate our approach with an industrial example of developing a CBTC train control system. We anticipate theory instantiation to be a promising direction for reusing theories via abstraction.

Electrospray mass spectrometry (ESI-MS) was used to measure the masses of an intact dimeric monoclonal antibody (Mab) and assess the fucosylation level. The Mab under study was EG2-hFc, a chimeric human-camelid antibody of about 80 kDa (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90). It was obtained from cell culture with and without a fucosylation inhibitor, and treated with EndoS which cleaves between the two core N-acetyl glucosamine (GlcNAc) residues. It is the first time that this combined approach with a unique mass spectrometer was used to measure 146 Da differences as part of a large intact dimeric antibody. Results showed that in the dimer, both heavy chains were fucosylated on the core GlcNAc of the Fc Asn site equivalent to Asn297. In the presence of the fucosylation inhibitor, fucosylation was lost on both subunits. Following reduction, monomers were analyzed and the masses obtained corroborated the dimer results. Dimeric EG2-hFc Mab treated with PNGase F, to deglycosylate the protein, was also measured by MS for mass comparison. In spite of the success of fucosylation level measurements, the experimental masses of deglycosylated dimers and GlcNAc-Fuc bearing dimers did not correspond to masses of our sequence of reference (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90; ; ), which prompted experiments to determine the protein backbone sequence. Digest mixtures from trypsin, GluC, as well as trypsin + GluC proteolysis were analyzed by matrix-assisted laser desorption/ionization (MALDI) MS and MS/MS. A few variations were found relative to the reference sequence, which are discussed in detail herein. These measurements allowed us to build a new "experimental" sequence for the EG2-hFc samples investigated in this work, although there are still ambiguities to be resolved in this new sequence. MALDI-MS/MS also confirmed the fucosylation pattern in the Fc tryptic peptide EEQYNSTYR.

Summary Background Catheter-based renal denervation has significantly reduced blood pressure in previous studies. Following a positive pilot trial, the SPYRAL HTN-OFF MED (SPYRAL Pivotal) trial was designed to assess the efficacy of renal denervation in the absence of antihypertensive medications. Methods In this international, prospective, single-blinded, sham-controlled trial, done at 44 study sites in Australia, Austria, Canada, Germany, Greece, Ireland, Japan, the UK, and the USA, hypertensive patients with office systolic blood pressure of 150 mm Hg to less than 180 mm Hg were randomly assigned 1:1 to either a renal denervation or sham procedure. The primary efficacy endpoint was baseline-adjusted change in 24-h systolic blood pressure and the secondary efficacy endpoint was baseline-adjusted change in office systolic blood pressure from baseline to 3 months after the procedure. We used a Bayesian design with an informative prior, so the primary analysis combines evidence from the pilot and Pivotal trials. The primary efficacy and safety analyses were done in the intention-to-treat population. This trial is registered at ClinicalTrials.gov , NCT02439749 . Findings From June 25, 2015, to Oct 15, 2019, 331 patients were randomly assigned to either renal denervation (n=166) or a sham procedure (n=165). The primary and secondary efficacy endpoints were met, with posterior probability of superiority more than 0·999 for both. The treatment difference between the two groups for 24-h systolic blood pressure was −3·9 mm Hg (Bayesian 95% credible interval −6·2 to −1·6) and for office systolic blood pressure the difference was −6·5 mm Hg (−9·6 to −3·5). No major device-related or procedural-related safety events occurred up to 3 months. Interpretation SPYRAL Pivotal showed the superiority of catheter-based renal denervation compared with a sham procedure to safely lower blood pressure in the absence of antihypertensive medications. Funding Medtronic.

Background Over 28 000 individuals were infected with Ebola virus during the West Africa (2013–2016) epidemic, yet there has been criticism of the lack of robust clinical descriptions of Ebola virus disease (EVD) illness from that outbreak. Objectives To perform a meta-analysis of published data from the epidemic to describe the clinical presentation, evolution of disease, and predictors of mortality in individuals with EVD. To assess the quality and utility of published data for clinical and public health decision-making. Data sources Primary articles available in PubMed and published between January 2014 and May 2017. Eligibility Studies that sequentially enrolled individuals hospitalized for EVD and that reported acute clinical outcomes. Methods We performed meta-analyses using random-effect models and assessed heterogeneity using the I2 method. We assessed data representativeness by comparing meta-analysis estimates with WHO aggregate data. We examined data utility by examining the availability and compatibility of data sets. Results In all, 3653 articles were screened and 34 articles were included, representing 16 independent cohorts of patients (18 overlapping cohorts) and at least 6168 individuals. The pooled estimate for case fatality rate was 51% (95% CI 46%–56%). However, pooling of estimates for clinical presentation, progression, and predictors of mortality in individuals with EVD were hampered by significant heterogeneity, and inadequate data on clinical progression. Our assessment of data quality found that heterogeneity was largely unexplained, and data availability and compatibility were poor. Conclusions We have quantified a missed opportunity to generate reliable estimates of the clinical manifestations of EVD during the West Africa epidemic. Clinical data standards and data capture platforms are urgently needed.

The B-Method has an interesting history, where language and tools have evolved over the years. This not only led to considerable research and progress in the area of formal methods, but also to numerous industrial applications, in particular in the railway domain. We present a survey of the industrial usage of the B-Method since the first toolset in 1993 and the inauguration of the driverless metro line 14 in Paris in 1999. We discuss the various areas of applications, from software development to data validation and on to systems modelling. The evolution of the tooling landscape is also analysed, and we present an assessment of the current situation, lessons learned and possible new directions.

Made available in DSpace on 2020-12-12T01:32:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification. Science and Technology Institute Federal University of São Paulo (UNIFESP) Viral Immunology Laboratory Butantan Institute Medical School São Paulo State University (UNESP) National Institute for Biotechnology Research and Training (NIBRT) Medical School São Paulo State University (UNESP)

The manufacturing of recombinant protein is traditionally undertaken in mammalian cell culture. Today, speed, cost and safety are the primary considerations for process improvements in both upstream and downstream manufacturing. Leaders in the biopharmaceutical industry are striving for continuous improvements to increase throughput, lower costs and produce safer more efficacious drugs. This can be achieved through advances in cell line engineering, process development of cell culture, development of chemically defined media and increased emphasis on product characterization. In the first part, this review provides a historical perspective on approved biotherapeutics by regulatory bodies which pave the way for next-generation products (including gene therapy). In the second part, it focuses on the application of in vitro and in vivo cell line engineering approaches, modern process development improvements including continuous manufacturing, recent developments in media formulation, and improvements in critical quality attribute determinations for products produced predominantly in mammalian cells.

Although popular in industry, state-chart notations with ‘run to completion’ semantics lack formal refinement and rigorous verification methods. State-chart models are typically used to design complex control systems that respond to environmental triggers with a sequential process. The model is usually constructed at a concrete level and verified and validated using animation techniques relying on human judgement. Event-B, on the other hand, is based on refinement from an initial abstraction and is designed to make formal verification by automatic theorem provers feasible. We introduce a notion of refinement into a ‘run to completion’ statechart modelling notation, and leverage Event-B ’s tool support for theorem proving. We describe the difficulties in translating ‘run to completion’ semantics into Event-B refinements and suggest a solution. We illustrate our approach and show how critical (e.g. safety) invariant properties can be verified by proof despite the reactive nature of the system. We also show how behavioural aspects of the system can be verified by testing the expected reactions using a temporal logic model checking approach.

Cross-layer runtime management (RTM) frameworks for embedded systems provide a set of standard APIs for communication between different system layers (i.e. RTM, applications and device) and simplify the development process by abstracting these layers. Integration of independently developed components of the system is an error-prone process that requires careful verification. In this paper, we propose a formal approach to integration testing through automatic generation of runtime assertions in order to test the implementation of the APIs. Our approach involves a formal model of the APIs, developed using the Event-B formal method which is automatically translated to a set of assertions and embedded in the existing implementation of APIs. The embedded assertions are used at runtime to check the correctness of the integration. This work was supported by the EPSRC PRiME Project (EP/K034448/1), www.prime-project.org.

This chapter presents the results of the railway domain. It focuses on the validation of a route control system using a model-based approach and formal methods. A main technical challenge is the automation of the translation step between a formalized specification and a mathematical model which allows for proofs of functional and safety properties of the solution.

IntroductionRates of chronic pain in military personnel are disproportionately high. Chronic pain is often associated with mental health and substance use disorders as comorbid conditions, making treatment of chronic pain complex. Mindfulness-based interventions (MBIs) are a promising behavioral approach to managing chronic pain and psychosocial sequelae. The unique nature of the military context may require adaptations to original MBIs for successful delivery in active-duty military populations. This study adapted the mindfulness-based stress reduction (MBSR) program to create a mindfulness training program that was relevant to active-duty Army personnel experiencing chronic pain. This article delineates the adaptation process employed to modify the MBSR program to the military context and discusses the resulting training program.Materials and MethodsThe adaptation process consisted of three iterative stages: 1) Drafting the preliminary intervention protocol with recommendations from stakeholders, including military healthcare providers; 2) Refining the preliminary protocol after pretesting the sessions with research team members and a military Veteran advisory committee; and 3) Delivering the preliminary protocol to one cohort of active-duty Soldiers with chronic pain, collecting feedback, and further refining the intervention protocol.ResultsMilitary-related adaptations to MBSR addressed three areas: military culture, language and terminology, and practical and logistical factors relevant to implementation in the military setting. This adaptation process resulted in a live, online program with six, weekly, sessions. Feedback from a military Veteran advisory committee resulted in modifications, including increasing military-relevant examples; preliminary testing with the target population resulted in additional modifications, including shortening the sessions to 75 min and structuring discussions more efficiently.ConclusionsThe adaptation process was successful in generating an engaging mindfulness training program that was highly relevant to the military context. Obtaining input from stakeholders, such as military healthcare providers and active-duty soldiers, and iterative feedback and modification, were key to the process. Moreover, the program was designed to maintain the integrity and core elements of MBIs while adapting to military culture. A future randomized controlled trial design will be used to evaluate the effectiveness of the intervention in improving chronic pain in military personnel. This program is responsive to the military’s call for nonpharmacologic treatments for chronic pain that are easily accessible. If effective, the mindfulness program has the potential for widespread dissemination to complement standard care for Service Members experiencing chronic pain.

In this work, we present an optical transit DEP flow cytometer for parallel single-cell analysis. Each cell's dielectric property is inferred from velocity perturbations due to DEP actuation in a microfluidic channel. Dual LED sources facilitate velocity measurement by producing two transit shadows for each cell passing through the channel. These shadows are detected using a 256-pixel linear optical array detector. Massively parallel analysis is possible as each pixel of the detector can independently analyze the passing cells. A wide channel (∼18 mm) was employed to carry many particles simultaneously, and the system was capable of detecting the velocity of over 200 cells simultaneously. We have achieved analysis rates for 10 µm diameter polystyrene spheres response exceeding 250 per second. With appropriate calibration, this DEP cytometer can quantitatively measure the dielectric response. The dielectric response (Clausius-Mossotti factor) of viable CHO cells was measured over the frequency range of 100 kHz to 6 MHz, and the obtained response matches the previously measured values by our group. The DEP cytometer uses simple modular components to achieve high throughput label-free single-cell dielectric analysis and can begin analyzing particles within 10 s after starting to pump the sample into the channel.

The dielectric properties of biological cells can be used to gain information on their physiology and morphology. We present a wideband dielectrophoresis (DEP) sensor that measures the dielectric spectrum of single cells while they flow through a microfluidic channel. We describe how an asymmetric co-planar electrode array can be used to apply DEP actuation to individual cells and then accurately measure their translation. This enables determination of the equivalent complex dielectric permittivity of the cell. Measurement over the beta-dispersion (interfacial) frequency range then facilitates determination of the cell's structural dimensions and the dielectric properties of its compartments. We measure the dielectric spectrums of viable and non-viable Chinese hamster ovary (CHO) cells, as induced through starvation, and show how they are related to changes in the cell’s membrane permittivity and cytoplasm conductivity.

The Event-B formalism allows program specifications to be modelled at an abstract level and refined towards a concrete model. However, Event-B lacks explicit control flow structure and ordering is implicitly encoded in event guards. This makes it difficult to identify and apply rules for transformation of Event-B models to sequential code. This paper introduces a scheduling language to support the incremental derivation of algorithmic control structure for events as part of the Event-B refinement process. We provide intermediate control structures for non-deterministic iteration and choice that ease the transition from abstract specifications to sequential implementations. We present rules for transforming algorithmic structures to more concrete refinements. We illustrate our approach by applying our method to the Schorr–Waite graph marking algorithm.

Humans infected with H7N9 avian influenza viruses can result in severe pneumonia and acute respiratory syndrome with an approximately 40% mortality rate, and there is an urgent need to develop an effective vaccine to reduce its pandemic potential. In this study, we used a novel PELC/CpG adjuvant for recombinant H7HA (rH7HA) subunit vaccine development. After immunizing BALB/c mice intramuscularly, rH7HA proteins formulated in this adjuvant instead of an alum adjuvant elicited higher IgG, hemagglutination-inhibition, and virus neutralizing antibodies in sera; induced higher numbers of H7HA-specific IFN-γ-secreting T cells and antibody secreting cells in spleen; and provided improved protection against live virus challenges. Our results indicate that rH7HA proteins formulated in PELC/CpG adjuvant can induce potent anti-H7N9 immunity that may provide useful information for H7N9 subunit vaccine development.

An integrated dielectrophoresis cytometer has been designed and implemented using a standard 0.35 μm CMOS technology. This design makes use of differential ring oscillators. The frequency difference between the two oscillators, in presence of a cell, is detected by an XOR gate. The frequency difference is then measured by a frequency counter. This sensor has ∼14 aF capacitance sensitivity at ∼1.4 GHz. It is used for detection of 10–20 μm polystyrene spheres and Chinese Hamster Ovary cells using coplanar microelectrodes on top of the CMOS chip. The design demonstrates that it is possible to differentiate between particles experiencing an altitude change resulting from either a positive or negative dielectrophoretic force in a microfluidic channel. The total area occupied by the DEP system is 0.6 mm2. This sensor can be used in high throughput applications with a density of more than 160 DEP sensor units in 1 cm2. The sensor integrates all the required parts of a DEP cytometer -excluding the pumping system and frequency counter-, making a semi-integrated lab on chip system for single cell detection and analysis.

Human interferon gamma (hIFNγ) affects tumour cells and modulates immune responses, showing promise as an anti-cancer biotherapeutic. This study investigated the effect of glycosylation and expression system of recombinant hIFNγ in ovarian carcinoma cell lines, PEO1 and SKOV3. The efficacy of E. coli- and mammalian-expressed hIFNγ (hIFNγ-CHO and HEK293, glycosylated/de-glycosylated) on cytostasis, cell death (MTT, and Guava-ViaCount® flow-cytometry) and apoptotic signalling (Western blot of Cdk2, histone H3, procaspase-3, FADD, cleaved PARP, and caspase-3) was examined. Hydrophilic Interaction Liquid Chromatography determined the structure of N-linked glycans present in HEK293-expressed hIFNγ (hIFNγ-HEK). PEO1 was more sensitive to hIFNγ than SKOV3, but responses were dose-dependent and expression platform/glycosylation status-independent, whereas SKOV3 responded to mammalian-expressed hIFNγ in a dose-independent manner, only. Complex-type oligosaccharides dominated the N-glycosylation pattern of hIFNγ-HEK with some terminal sialylation and core fucosylation. Cleaved PARP and cleaved caspase-3 were not detected in either cell line, but FADD was expressed in SKOV3 with levels increased following treatment. In conclusion, hIFNγ did not induce apoptosis in either cell line. Mammalian- expressed hIFNγ increased cell death in the drug-resistant SKOV3. The presence of FADD in SKOV3, which may inhibit apoptosis through activation of NF-κB, could serve as a novel therapeutic target.

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

A key risk with autonomous systems (AS) is the trustworthiness of the decision-making and control mechanisms that replace human control. To be trustworthy, systems need to remain safe while being resilient to unpredictable changes, functional/operational failures and cybersecurity threats. Rigorous validation and verification are essential to ensure trustworthiness of AS. Current engineering practice relies heavily on Verification and Validation (V&V) test-and-fix of system characteristics which is very time-consuming and expensive, limiting the possibilities for exploration of alternatives in system design. Instead, we focus on building a correct-by-construction system. In this paper, we present an approach to identifying and analysing mission requirements for squads of autonomous missions. Clear definition of requirements is an important pre-requisite for mission planning and for V&V of mission management. We use a structured approach to requirements identification and use formal modelling to help remove ambiguities in the requirements and to specify formal properties that should be satisfied by the missions. Our approach use a combination of analysis techniques based on Systems Theoretic Process Analysis (STPA) and formal modelling to generate critical requirements that ensure the safety and security of the system. We also suggest a compositional approach for formal modelling to enhance re-usability and address the complexity of formal modelling. Our approach is being evaluated through consideration of a combined mission of an Unmanned Surface Vehicle (USV) with deployment/recovery of small Unmanned Underwater Vehicles (UUV) within a shipping channel whereby the USV has to safely maintain station for a long period and then proceed to recover the UUV, while maintaining a communication link to an Unmanned Aerial Vehicle (UAV).

TAXPAYER confidentiality has a long history of protection in the UK. It is a fundamental part of the tax system. It has been considered invaluable by the executive for the efficient collection of taxation, protected by Parliament since the Income Tax Act 1799 and recognised by the courts as a “vital element in the working of the system” (Inland Revenue Commissioners v National Federation of Self-Employed and Small Businesses Ltd. [1982] A.C. 617, 633, per Lord Wilberforce).

BACKGROUND: While cardiac rehabilitation (CR) is recommended for all patients after an acute cardiac event, limitations exist in reach. OBJECTIVE: The purpose of the current study was to develop and pilot a flexible online CR program based on self-management principles "Help Yourself Online." METHODS: The program was designed as an alternative to group-based CR as well as to complement traditional CR. The program was based on existing self-management resources developed previously by the Heart Research Centre. Twenty-one patients admitted to Cabrini Health for an acute cardiac event were recruited to test the program. The program was evaluated using qualitative and quantitative methods. RESULTS: Quantitative results demonstrated that patients believed the program would assist them in their self-management. Qualitative evaluation, using focus group and interview methods with 15 patients, showed that patients perceived the online CR approach to be a useful instrument for self-management. CONCLUSIONS: Broader implications of the data include the acceptability of the intervention, timing of intervention delivery, and patients' desire for additional online community support.

Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity are well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analyzed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80 kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150 kDa). This was achieved by the addition of three glycosylation inhibitors (swainsonine, castanospermine, and kifunensine) independently to Chinese hamster ovary (CHO) cell cultures to generate hybrid and high mannose glycan structures. Biophysical analysis by circular dichroism, dynamic light scattering and analytical ultra-centrifugation confirmed that the solution-behaviour of the mAbs remained constant over multiple concentrations and glycan treatments. However, changes were observed when studying the interaction of FcγRI with variously glycosylated mAbs. Both mAbs were observed to have a decreased binding affinity upon treatment with swainsonine which produced hybrid glycans. Following de-glycosylation the binding affinity for EG2-hFc was only marginally reduced (6-fold) compared to a drastic (118-fold) decrease for DP-12. In summary, our data suggest that the relatively low molecular weight of chimeric EG2-hFc may contribute to its enhanced stability against glycan changes making it a highly suitable mAb candidate for therapeutic applications.

Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- fluoro peracetylated fucose (2FF), which was added to cell cultures expressing the EG2-hFc antibody in increasing concentrations up to 50μM. At this concentration there was no observed effect on cell growth. Glycan analysis was performed on antibodies collected from culture samples using HILIC-HPLC. The inhibitor reduced total fucosylation from 80% to 17.5% at 20μM 2FF. The second method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de novo pathway into producing rhamnose which is not incorporated into a glycan. Stable clones from transfected pools were isolated following flow cytometry using the green fluorescent protein (GFP) marker which was co-expressed with the RMD gene. High expressing RMD clones reduced the fucosylation of the antibody glycan to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the fucosylation level even further to 3% of the antibody glycan. An incremental increase in fucosylation was obtained by step-wise addition of fucose (up to 1 mM) to the RMD cells, in which the fucosylation level increased to a maximum of 87%. We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with addition of increasing concentrations of 2FF. This showed that 2FF inhibits the addition of fucose in a concentration- dependent and specific manner with the inhibition of fucose occurring one fucose at a time. Control cultures showed the presence of a predominant peak indicating two fucose moieties per antibody. As the 2FF inhibitor concentration was increased peaks corresponding to one fucose per antibody and non-fucosylated antibody predominated with a gradual decrease of the 2 fucose peak to insignificance at 15 μM 2FF.

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed-batch cultures. To study amino acid limitation effects, a serum-free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine, and cysteine on the cell growth, metabolism, antibody productivity, and product glycosylation was investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine limitation was detrimental to both cell proliferation and productivity for all three CHO cell lines. Glutamine limitation reduced growth but not cell specific productivity, whereas asparagine limitation had no significant effect on either growth or cell specific productivity. Neither glutamine nor asparagine limitation significantly affected antibody glycosylation. Replenishing the CHO-DXB11 culture with cysteine after 1 day of cysteine limitation allowed the cells to partially recover their growth and productivity. This recovery was not observed after 2 days of cysteine limitation. Based on these findings, a fed-batch protocol was developed using single or mixed amino acid supplementation. Although cell density and antibody concentration were lower compared to a commercial feed, the feeds based on cysteine supplementation yielded comparable cell specific productivity. Overall, this study showed that different amino acid limitations have varied effects on the performance of CHO cell cultures and that maintaining cysteine availability is a critical process parameter for the three cell lines investigated.

The increased connectivity of critical maritime infrastructure (CMI) systems to digital networks have raised concerns of their vulnerability to cyber attacks. As less emphasis has been placed, to-date, on ensuring security of cyber-physical maritime systems, mitigating these cyber attacks will require the design and engineering of secure maritime infrastructure systems. Systems theory has been shown to provide the foundation for a disciplined approach to engineering secure cyber-physical systems. In this paper, we use systems theory, and concepts adapted from safety analysis, to develop a systematic mechanism for analysing the security functionalities of assets’ interactions in the maritime domain. We use the theory to guide us to discern the system’s requirement, likely system losses, potential threats, and to construct system constraints needed to inhibit or mitigate these threats. Our analyses can be used as springboards to a set of principles to help enunciate the assumptions and system-level security requirements useful as the bases for systems’ security validation and verification.

Behaviour driven formal model development (BDFMD) enables domain engineers to influence and validate mathematically precise and verified specifications. In previous work we proposed a process where manually authored scenarios are used initially to support the requirements and help the modeller. The same scenarios are used to verify behavioural properties of the model. The model is then mutated to automatically generate scenarios that have a more complete coverage than the manual ones. These automatically generated scenarios are used to animate the model in a final acceptance stage. In this paper, we discuss lessons learned from applying this BDFMD process to a real-life specification: The European Train Control Systems (ETCS) Hybrid Level 3. During the case study, we have developed our understanding of the process, modifying the way we do some stages and developing improved tool support to make the process more efficient. We discuss (1) the need for abstract scenarios during incremental model development and verification, (2) tools and techniques developed to make the running of scenarios more efficient, and (3) improvements to tools that generate new test cases to improve coverage.

ion and refinement offer a stepwise development approach to managing complexity in system design. Based on our previous work that extends Event-B models with high level real-time trigger-response properties, this paper presents refinement semantics of timed systems using behavioral traces. Forward simulation, which is a proof technique for refinement, is used to verify the consistency between different refinement levels. To prove refinement of trace semantics, we construct intermediate traces from concrete traces with a mapping function and prove the intermediate trace without stuttering events and states are abstract traces. Fairness assumptions, relative deadlock freedom, and conditional convergence are adopted in refinement steps to eliminate Zeno behavior in timed models. Based on the semantics, we develop refinement rules and strategies to perform refinement on timed models and refine real-time trigger-response properties into sequential or alternative sub-timing properties with proofs.