Developing a lactate-inducible transgene expression system for use in Chinese hamster ovary cells

The accumulation of lactate during cultivation of mammalian cells for biopharmaceutical production is a longstanding issue affecting glycosylation quality and productivity. Many approaches exist to mitigate its impact, either through the replacement of glucose with slowly metabolised sugars, dynamic feeding strategies, or host cell engineering. The manipulation of genes in this latter approach is constitutive and may suboptimally respond to cellular needs. The LldR proteins from Corynebacterium glutamicum and Pseudomonas aeruginosa have been used in this project to create a lactate-inducible transgene expression system, which can be used subsequently to dynamically drive expression of proteins previously targeted to mitigate the accumulation of lactate. Expression and purification of these LldR proteins, fused to transcriptional effector domains in various orientations and with fusion linkers in certain cases, allowed in vitro characterisation and optimisation of the constituent parts of the inducible system. This provided crucial information in some cases about the need to use a flexible linker between LldR and a VP64 transactivation domain. In vivo experimentation of these optimised systems showed significant levels of induction in response to 20 mM lactate, with a 3.46-fold decrease in expression seen for one construct. Some preliminary work was also carried out with Cas9-VPR, which was shown to be able to upregulate transiently transfected genes up to 1.6-fold. In the future, this will be a useful tool for upregulating multiple previously identified targets, as well as helping to find new beneficial targets. The general approach outlined here for the development of this lactate-inducible transgene expression system will be appropriate for any other such project where the ligand of interest is a central metabolite. Inherently weaker induction might be a feature of such a system, given the presence of the inducer at low and relatively benign or neutral concentrations throughout a period of interest; testing an unoptimised system in mammalian cells may return little or no detectable induction signal and therefore it will not be straightforward to optimise such a system solely through the use of in vivo experimentation. In vitro characterisation of the inducible system components, as performed here, can provide essential feedback regarding the impact of effector domain fusion and operator design on the DNA-binding affinity of the biosensor prior to in vivo testing. The work in this thesis will allow the future exploration of dynamically regulated host cell engineering designed to combat the lactate accumulation phenotype.Open Acces

A Study of the Feasibility and Potential Implementation of Metro-Based Freight Transportation in Newcastle upon Tyne

The concept of using a metropolitan railway network to transport freight directly to a city centre from the surrounding businesses has been the subject of much research. This paper looks in depth at the Tyne and Wear Metro system, situated in Newcastle upon Tyne, to determine if such a scheme would be feasible. Through research into the modes of transport available, along with a review of literature and case studies, it was found that the current method of transporting the majority of freight by road is unsustainable and damaging to both the environment and local communities. Other options for the transportation of freight have been reviewed, and results showed that a modal shift will be necessary in the near future. The system was then modelled using software provided by the Department for Transport, which demonstrated that the implementation of such a scheme would provide vast accident savings, a reduction in the number of casualties on the road, and a monetary saving as a result of the lower casualty rate. The conclusion was reached that the scheme is viable, however further research and study is necessary before implementation

Assessing frailty amongst older people admitted to hospital in a low-income setting: a multicentre study in northern Tanzania

\ua9 The Author(s) 2024.Background: Populations are ageing globally and Low- and Middle-Income Countries (LMICs) are experiencing the fastest rates of demographic change. Few studies have explored the burden of frailty amongst older people in hospital in LMICs, where healthcare services are having to rapidly adapt to align with the needs of older people. This study aimed to measure the prevalence of frailty amongst older people admitted to hospital in Tanzania and to explore their demographic and clinical characteristics. Methods: This study had a prospective observational design. Over a six-month period, all adults ≥ 60 years old admitted to medical wards in four hospitals in northern Tanzania were invited to participate. They were screened for frailty using the Clinical Frailty Scale (CFS) and the Frailty Phenotype (FP). Demographic and clinical characteristics of interest were recorded in a structured questionnaire. These included the Barthel Index, the Identification of Elderly Africans Instrumental Activities of Daily Living (IADEA-IADL) and Cognitive (IDEA-Cog) screens, the EURO-D depression scale and Confusion Assessment Method. Results: 540 adults aged ≥ 60 were admitted, and 308 completed assessment. Frailty was present in 66.6% using the CFS and participants with frailty were significantly older, with lower levels of education and literacy, greater disability, greater comorbidity, poorer cognition and higher levels of delirium. Using the FP, 57.0% of participants were classed as frail though a majority of participants (n = 159, 51.6%) could not be classified due to a high proportion of missing data. Conclusions: This study indicates that the prevalence of frailty on medical wards in northern Tanzania is high according to the CFS. However, the challenges in operationalising the FP in this setting highlight the need for future work to adapt frailty screening tools for an African context. Future investigations should also seek to correlate frailty status with long-term clinical outcomes after admission in this setting

A Check-in and Bag Drop Service On-board Light Rail Vehicles for Passengers Travelling to the Airport

It appears that nowadays rail vehicles are not the primary choice of transportation for people going to the airport. The inconvenience of carrying luggage on railways deters passengers, who look for alternatives. Attempts have been made to encourage passengers to travel to the airport by rail. However, significant limitations in these existing systems suggest a need for extensive work and adjustments, but this would increase the price and discourage passengers. This study investigates the potential for implementing an on-board check-in and bag drop system onto rail vehicles. By observing the Tyne and Wear Metro, Newcastle, the UK the benefits and limitations of installing such a facility have been explored, by the development of suitable operations and interior designs. Four designs which meet the design criteria were produced and their limitations considered. This study concludes that the potential for an on-board check-in and bag drop facility is realistic. Each design brings key benefits and limitations, and all meet security, and health and safety criteria. A feature incorporated into all designs allows for the equipment to be removed easily and stored away, helping with a low cost and versatile approach

Evaluating the Benefits of Aphasia Intervention Delivered in Virtual Reality: Results of a Quasi-Randomised Study

Introduction This study evaluated an intervention for people with aphasia delivered in a novel virtual reality platform called EVA Park. EVA Park contains a number of functional and fantastic locations and allows for interactive communication between multiple users. Twenty people with aphasia had 5 weeks’ intervention, during which they received daily language stimulation sessions in EVA Park from a support worker. The study employed a quasi randomised design, which compared a group that received immediate intervention with a waitlist control group. Outcome measures explored the effects of intervention on communication and language skills, communicative confidence and feelings of social isolation. Compliance with the intervention was also explored through attrition and usage data. Results There was excellent compliance with the intervention, with no participants lost to follow up and most (18/20) receiving at least 88% of the intended treatment dose. Intervention brought about significant gains on a measure of functional communication. Gains were achieved by both groups of participants, once intervention was received, and were well maintained. Changes on the measures of communicative confidence and feelings of social isolation were not achieved. Results are discussed with reference to previous aphasia therapy findings

PASIV - A pooled approach-based workflow to overcome toxicity-induced Design of Experiments failures and inefficiencies

We present here a newly developed workflow - which we have called PASIV - designed to provide a solution to a practical problem with Design of Experiments methodology (DoE): i.e. what can be done if the scoping phase of the DoE cycle is severely hampered by burden and toxicity issues (either caused by the metabolite or an intermediary) making it unreliable or impossible to proceed to the screening phase? PASIV - standing for Pooled Approach, Screening, Identification and Visualization - was designed so the (viable) region of interest can be made to appear through an interplay between biology and software. This was achieved by combining multiplex construction in a pooled approach (one pot reaction) with a viability assay, and with a range of bioinformatic tools (including a novel construct matching tool). PASIV was tested on the exemplar of the lycopene pathway - under stressful constitutive expression yielding a region of interest with comparatively stronger producers

Removing the bottleneck : introducing cMatch - a lightweight tool for construct-matching in synthetic biology

We present a software tool, called cMatch, to reconstruct and identify synthetic genetic constructs from their sequences, or a set of sub-sequences - based on two practical pieces of information: their modular structure, and libraries of components. Although developed for combinatorial pathway engineering problems and addressing their quality control (QC) bottleneck, cMatch is not restricted to these applications. QC takes place post assembly, transformation and growth. It has a simple goal, to verify that the genetic material contained in a cell matches what was intended to be built - and when it is not the case, to locate the discrepancies and estimate their severity. In terms ofreproducibility/reliability, the QC step is crucial. Failure at this step requires repetition of the construction and/or sequencing steps. When performed manually or semi-manually QC is an extremely time-consuming, error prone process, which scales very poorly with the number of constructs and their complexity. To make QC frictionless and more reliable, cMatch performs an operation we have called ‘construct-matching’ and automates it. Construct-matching is more thorough than simple sequence-matching, as it matches at the functional level-and quantifies the matching at the individual component level and across thewhole construct