Production of a high purity, C-tagged hepatitis B surface antigen fusion protein VLP vaccine for malaria expressed in Pichia pastoris under cGMP conditions.

Virus-like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP-based anti-sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical-grade material for use in human clinical trials. The R21 construct was re-engineered to include a C-tag to allow affinity-based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C-tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high-level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C-tag platform technologies to enable cGMP-compliant biomanufacturing of high purity yeast-expressed VLP-based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame.

Microneedle-based devices for point-of-care infectious disease diagnostics.

Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis.

Theranostic Near-Infrared-Active Conjugated Polymer Nanoparticles.

Conjugated polymer nanoparticles (CPNs) based on a common solar cell material (PTB7) have been prepared, and their potential in theranostic applications based on bioimaging and photosensitizing capabilities has been evaluated. The main absorption and emission bands of the prepared CPNs both fell within the NIR-I (650-950 nm) transparency window, allowing facile and efficient implementation of our CPNs as bioimaging agents, as demonstrated in this work for A549 human lung cancer cell cultures. The prepared CPN samples were also shown to produce reactive oxygen species (ROS) upon photoexcitation in the near-infrared or ultraviolet spectral regions, both in aqueous solutions and in HaCaT keratinocyte cell cultures. Importantly, we show that the photosensitizing ability of our CPNs was largely determined by the nature of the stabilizing shell: coating the CPNs with a Pluronic F-127 copolymer led to an improvement of photoinitiated ROS production, while using poly[styrene-co-maleic anhydride] instead completely quenched said process. This work therefore demonstrates that the photosensitizing capability of CPNs can be modulated via an appropriate selection of stabilizing material and highlights the significance of this parameter for the on-demand design of theranostic probes based on CPNs.

Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called "RH5.1" was produced as a soluble product under cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a high-producing monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at -80 °C and is stable for over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2 cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and "difficult-to-express" recombinant protein-based vaccines.

Correction: Cellular bioenergetics is impaired in patients with chronic fatigue syndrome.

[This corrects the article DOI: 10.1371/journal.pone.0186802.].

Cellular bioenergetics is impaired in patients with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a highly debilitating disease of unknown aetiology. Abnormalities in bioenergetic function have been cited as one possible cause for CFS. Preliminary studies were performed to investigate cellular bioenergetic abnormalities in CFS patients. A series of assays were conducted using peripheral blood mononuclear cells (PBMCs) from CFS patients and healthy controls. These experiments investigated cellular patterns in oxidative phosphorylation (OXPHOS) and glycolysis. Results showed consistently lower measures of OXPHOS parameters in PBMCs taken from CFS patients compared with healthy controls. Seven key parameters of OXPHOS were calculated: basal respiration, ATP production, proton leak, maximal respiration, reserve capacity, non-mitochondrial respiration, and coupling efficiency. While many of the parameters differed between the CFS and control cohorts, maximal respiration was determined to be the key parameter in mitochondrial function to differ between CFS and control PBMCs due to the consistency of its impairment in CFS patients found throughout the study (p≤0.003). The lower maximal respiration in CFS PBMCs suggests that when the cells experience physiological stress they are less able to elevate their respiration rate to compensate for the increase in stress and are unable to fulfil cellular energy demands. The metabolic differences discovered highlight the inability of CFS patient PBMCs to fulfil cellular energetic demands both under basal conditions and when mitochondria are stressed during periods of high metabolic demand.

Investigating the running abilities of Tyrannosaurus rex using stress-constrained multibody dynamic analysis.

The running ability of Tyrannosaurus rex has been intensively studied due to its relevance to interpretations of feeding behaviour and the biomechanics of scaling in giant predatory dinosaurs. Different studies using differing methodologies have produced a very wide range of top speed estimates and there is therefore a need to develop techniques that can improve these predictions. Here we present a new approach that combines two separate biomechanical techniques (multibody dynamic analysis and skeletal stress analysis) to demonstrate that true running gaits would probably lead to unacceptably high skeletal loads in T. rex. Combining these two approaches reduces the high-level of uncertainty in previous predictions associated with unknown soft tissue parameters in dinosaurs, and demonstrates that the relatively long limb segments of T. rex-long argued to indicate competent running ability-would actually have mechanically limited this species to walking gaits. Being limited to walking speeds contradicts arguments of high-speed pursuit predation for the largest bipedal dinosaurs like T. rex, and demonstrates the power of multiphysics approaches for locomotor reconstructions of extinct animals.

Understanding Muscle Dysfunction in Chronic Fatigue Syndrome.

Introduction. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a debilitating disorder of unknown aetiology, characterised by severe disabling fatigue in the absence of alternative diagnosis. Historically, there has been a tendency to draw psychological explanations for the origin of fatigue; however, this model is at odds with findings that fatigue and accompanying symptoms may be explained by central and peripheral pathophysiological mechanisms, including effects of the immune, oxidative, mitochondrial, and neuronal pathways. For example, patient descriptions of their fatigue regularly cite difficulty in maintaining muscle activity due to perceived lack of energy. This narrative review examined the literature for evidence of biochemical dysfunction in CFS/ME at the skeletal muscle level. Methods. Literature was examined following searches of PUB MED, MEDLINE, and Google Scholar, using key words such as CFS/ME, immune, autoimmune, mitochondria, muscle, and acidosis. Results. Studies show evidence for skeletal muscle biochemical abnormality in CFS/ME patients, particularly in relation to bioenergetic dysfunction. Discussion. Bioenergetic muscle dysfunction is evident in CFS/ME, with a tendency towards an overutilisation of the lactate dehydrogenase pathway following low-level exercise, in addition to slowed acid clearance after exercise. Potentially, these abnormalities may lead to the perception of severe fatigue in CFS/ME.

A role for human mitochondrial complex II in the production of reactive oxygen species in human skin.

The mitochondrial respiratory chain is a major generator of cellular oxidative stress, thought to be an underlying cause of the carcinogenic and ageing process in many tissues including skin. Previous studies of the relative contributions of the respiratory chain (RC) complexes I, II and III towards production of reactive oxygen species (ROS) have focussed on rat tissues and certainly not on human skin which is surprising as this tissue is regularly exposed to UVA in sunlight, a potent generator of cellular oxidative stress. In a novel approach we have used an array of established specific metabolic inhibitors and DHR123 fluorescence to study the relative roles of the mitochondrial RC complexes in cellular ROS production in 2 types of human skin cells. These include additional enhancement of ROS production by exposure to physiological levels of UVA. The effects within epidermal and dermal derived skin cells are compared to other tissue cell types as well as those harbouring a compromised mitochondrial status (Rho-zero A549). The results show that the complex II inhibitor, TTFA, was the only RC inhibitor to significantly increase UVA-induced ROS production in both skin cell types (P<0.05) suggesting that the role of human skin complex II in terms of influencing ROS production is more important than previously thought particularly in comparison to liver cells. Interestingly, two-fold greater maximal activity of complex II enzyme was observed in both skin cell types compared to liver (P<0.001). The activities of RC enzymes appear to decrease with increasing age and telomere length is correlated with ageing. Our study showed that the level of maximal complex II activity was higher in the MRC5/hTERT (human lung fibroblasts transfected with telomerase) cells than the corresponding wild type cells (P=0.0012) which can be considered (in terms of telomerase activity) as models of younger and older cells respectively.

More than one way of being a moa: differences in leg bone robustness map divergent evolutionary trajectories in Dinornithidae and Emeidae (Dinornithiformes).

The extinct moa of New Zealand included three families (Megalapterygidae; Dinornithidae; Emeidae) of flightless palaeognath bird, ranging in mass from <15 kg to >200 kg. They are perceived to have evolved extremely robust leg bones, yet current estimates of body mass have very wide confidence intervals. Without reliable estimators of mass, the extent to which dinornithid and emeid hindlimbs were more robust than modern species remains unclear. Using the convex hull volumetric-based method on CT-scanned skeletons, we estimate the mass of a female Dinornis robustus (Dinornithidae) at 196 kg (range 155-245 kg) and of a female Pachyornis australis (Emeidae) as 50 kg (range 33-68 kg). Finite element analysis of CT-scanned femora and tibiotarsi of two moa and six species of modern palaeognath showed that P. australis experienced the lowest values for stress under all loading conditions, confirming it to be highly robust. In contrast, stress values in the femur of D. robustus were similar to those of modern flightless birds, whereas the tibiotarsus experienced the highest level of stress of any palaeognath. We consider that these two families of Dinornithiformes diverged in their biomechanical responses to selection for robustness and mobility, and exaggerated hindlimb strength was not the only successful evolutionary pathway.

Real-time monitoring of superoxide generation and cytotoxicity in neuroblastoma mitochondria induced by 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline.

The potential neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) has recently been suggested to be a causative factor in the clinical development of parkinsonian symptoms after long-term exposure to precursor compounds such as the hypnotic chloral hydrate. TaClo is known to cause cell death in dopaminergic neuronal cells, however, the pathway and mechanisms remain undefined. This study reports for the first time that TaClo promotes cytotoxicity in SH-SY5Y neuroblastoma cells within 2 hours of initial exposure. TaClo also caused superoxide production from isolated mitochondria, which was comparable in response time and magnitude to production elicited by more established respiratory inhibitors such as rotenone and antimycin A. These findings present new evidence in support of TaClo-induced neuronal death via superoxide signalling and oxidative stress.

Electrochemical and optical sensing of reactive oxygen species: pathway to an integrated intracellular and extracellular measurement platform.

A comprehensive understanding of ROS (reactive oxygen species)-dependent cellular interaction requires the previously unmet ability to simultaneously monitor the intra- and extra-cellular environments. The present review assesses the potential of novel electrochemical and fluorescent-based nanosensor approaches to address the limitations of existing techniques for ROS analysis. Data generated by these new approaches have already contributed significantly to current understanding of the roles that these species play in various in vitro scenarios. However, integration of these novel approaches has the potential to offer, for the first time, the unparalleled ability to measure simultaneously and in real-time ROS flux in both the intra- and extra-cellular environments.

Implications of using the fluorescent probes, dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species.

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.

Direct, real-time monitoring of superoxide generation in isolated mitochondria.

Mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian cells. The generation of ROS underlies many physiological and pathophysiological processes that occur within cellular systems. Superoxide ([image omitted] ) is the proximal ROS generated during electron 'leakage' from the mitochondrial electron transport chain (mETC) and is known to be released at mitochondrial complex I and complex III. Monitoring mitochondrial [image omitted] production directly and in real-time offers the potential to improve understanding of the complex mechanisms involved during mitochondrial [image omitted] generation. This study reports the novel application of a cytochrome c functionalized amperometric sensor for monitoring [image omitted] generation in isolated mitochondrial fractions. The non-invasive sensor system described allowed a comparison of [image omitted] production following specific inhibition of complex I and complex III of the mETC to be made directly and in real-time.

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species.

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.

Setting the agenda: an analysis of negotiation strategies in clinical talk.

This article analyzes the process whereby physicians and patients set the agenda for medical interviews. Applying a conversation analytic perspective to the analysis of 22 videotapes of primary care interviews at a large, urban, teaching and research hospital, a 3-stage model is developed, consisting of (a) an opening sequence, (b) an initial statement of concerns by the patient, and (c) the negotiation process. The analysis illustrates the critical function of the opening verbal exchanges, showing how patient responses to the physician's first question and subsequent queries and summaries by the physician are intricately interwoven. The interaction at the very beginning of the interview is shown to significantly alter the ensuing interaction. The analysis provides a discursive framework for analyzing problematic communication during the primary care interview.

Cu/Zn superoxide dismutase (SOD1) mutations associated with familial amyotrophic lateral sclerosis (ALS) affect cellular free radical release in the presence of oxidative stress.

INTRODUCTION: The exact molecular mechanisms by which mutations in Cu/Zn superoxide dismutase (SOD1) cause motor neuron injury remain incompletely understood, though a body of evidence suggests that the mutant protein exerts a cell-specific toxic gain of function. The role of nitric oxide (NO) in SOD1-related motor neuron injury has been particularly controversial. Theoretically, there are arguments to suggest that NO may exert an important role in motor neuron injury, but there is relatively little direct experimental support for this hypothesis. OBJECTIVES: The present study aimed to examine further the potential role for NO in motor neuron injury caused by mutant SOD1. METHOD: We have generated a cellular model of familial amyotrophic lateral sclerosis (ALS) by stably transfecting NSC34 cells with one of three mutant forms of SOD1 (G93A, G37R, I113T). In the presence of mutant SOD1, NSC34 cells show increased cell death following oxidative stress induced by serum withdrawal. This model of motor neuron death involves cellular release of superoxide and NO radicals, which were directly measured in real time using microelectrode biosensors. RESULTS: The expression of both normal and mutant SOD1 decreased the measured extracellular superoxide release, but had divergent effects on the measured release of NO. Normal SOD1 increased the measured NO release, whereas cells expressing mutant SOD1 released less NO. Co-administration of two different nitric oxide synthase inhibitors (L-NAME and L-N-methyl arginine) did show some neuroprotective effect, but this was only partial, and the effect was more marked using nuclear integrity as a measure of cell viability, rather than MTT conversion. Cells expressing mutant SOD1 were, however, more sensitive to toxicity induced by extrinsic exposure to NO, using a slow-release NO donor. CONCLUSION: NO is likely to contribute to motor neuron injury, but this does not fully account for all the cellular toxic effects of mutant SOD1.

Sensor-based measurements of the role and interactions of free radicals in cellular systems.

Direct real-time electrochemical measurements have offered new insight into the importance of free radical interplay in a number of cell culture and in vivo models of neurodegenerative processes. This review highlights investigations carried out in this laboratory of real-time superoxide and nitric oxide free radical generation, and presents evidence of complex inter-relationships between these species. These include: a novel function for astrocytic nitric oxide synthase in controlling neuronal nitric oxide availability; and the demonstration that extracellular superoxide flux can lead to the generation of NO by glial cells. The possible consequences of these interactions are discussed.