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Objectives: The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains.Methods: A commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays.Results: We have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 10⁷ cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 10⁵ cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays.Conclusions: We have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis. [ABSTRACT FROM AUTHOR]

This article examines the suitability of two different materials which are black graphite carbon fiber and red pre-impregnated fiberglass from which to fabricate artificial dragonfly wing frames. These wings could be of use in Biomimetic Micro Aerial Vehicles (BMAV). BMAV are a new class of unmanned micro-sized air vehicles that mimic flying biological organisms. Insects, such as dragonflies, possess corrugated and complex vein structures that are difficult to mimic. Simplified dragonfly wing frames were fabricated from these materials and then a nano-composite film was adhered to them, which mimics the membrane of an actual dragonfly. Experimental analysis of these results showed that although black graphite carbon fiber and red pre-impregnated fiberglass offer some structural advantages, red pre-impregnated fiberglass was a less preferred option due to its warpage and shrinking effects. Black graphite carbon fiber with its high load bearing capability is a more suitable choice for consideration in future BMAV applications.

Abstract Background Scotland has a policy aimed at increasing physical activity levels in the population, but evidence on how to achieve this is still developing. Studies that focus on encouraging real world participants to start physical activity in their settings are needed. The Walking for Well-being in the West study was designed to assess the effectiveness of a pedometer-based walking programme in combination with physical activity consultation. The study was multi-disciplinary and based in the community. Walking for Well-being in the West investigated whether Scottish men and women, who were not achieving the current physical activity recommendation, increased and maintained walking behaviour over a 12 month period. This paper outlines the rationale and design of this innovative and pragmatic study. Methods Participants were randomised into two groups: Group 1: Intervention (pedometer-based walking programme combined with a series of physical activity consultations); Group 2: Waiting list control for 12 weeks (followed by minimal pedometer-based intervention). Physical activity (primary outcome) was measured using pedometer step counts (7 day) and the International Physical Activity Questionnaire (long version). Psychological processes were measured using questionnaires relating to the Transtheoretical Model of Behaviour Change, mood (Positive and Negative Affect Schedule) and quality of life (Euroqol EQ-5D instrument). Physiological measures included anthropometric and metabolic outcomes. Environmental influences were assessed subjectively (Neighbourhood Quality of Life Survey) and objectively (neighbourhood audit tool and GIS mapping). The qualitative evaluation employed observation, semi-structured interviews and focus groups. A supplementary study undertook an economic evaluation. Discussion Data analysis is on-going. Walking for Well-being in the West will demonstrate if a pedometer based walking programme, in combination with physical activity consultation results in a sustainable increase in walking behaviour in this sample of Scottish adults over a 12 month period. The study will examine the complex relationships between behavioural change, health consequences and the role of the environment, in conjunction with the cost effectiveness of this approach and a detailed insight into the participants' experiences of the intervention. Trial registration Current Controlled Trials ISRCTN88907382

Background: Many aspects of microbial dissemination appear to vary with host cholesterol levels. Since neonatal septicemia remains a leading cause of newborn admissions and mortality in resource-limited settings, the contribution of abnormal cholesterol levels in maternal and/or newborn blood to the risk of neonatal septicemia and outcome requires elucidation. We aim to determine a relationship between maternal serum and neonatal cord blood cholesterol levels and neonatal septicemia., Methods: This will be a mother-newborn pair cohort study. Approximately 353 pregnant women who are eligible and consent to participate in the study will have blood drawn for a lipid profile. Upon delivery, we will analyse the cord blood cholesterol of their newborns and follow them for 28 days to determine whether the infants develop clinical signs and symptoms suggestive of neonatal septicemia. Relative risk will be used to determine the association between cholesterol and newborn septicemia. Poisson regression will be used to estimate the relative risk (with 95% confidence intervals) of developing septicemia., Discussion: Findings from our study will contribute evidence to support the inclusion of lipid profile screening for pregnant women and newborns. Our study will determine whether newborns with abnormal cholesterol or those born to mothers with abnormal cholesterol will require rigorous follow-up in neonatal clinics., (© 2022. The Author(s).)

Half of all cancer patients receive radiotherapy, which makes a substantial contribution to their long-term disease control/cure. There are significant inter-patient differences in response, both in terms of efficacy and toxicity (frequently delayed onset) which are difficult to predict. With the introduction of technological improvements (e.g. stereotactic body radiotherapy and proton therapy) and development of combination therapies (e.g. radiotherapy and immune checkpoint inhibition), predictive biomarkers are needed even more. Whilst genomic studies have contributed significantly to predictions of response to anticancer therapy, there is no doubt that more information can be gathered from patient tissue samples. Patients are willing to donate their tissues to biobanks and wish them to be used as widely as possible for high-quality research. We report here a survey of the current practices in the UK from several groups collecting material from patients in radiotherapy trials and have identified barriers to collecting and sharing data and samples. We believe the current situation represents a significant missed opportunity to improve the personalisation of radiotherapy. We propose a greater involvement of patients and/or their advocates, a standardisation of the patient information leaflet, consent form content and data set, with easy linkage to clinical data, which would facilitate widespread sample and data discovery and availability to other researchers. The greater sharing of data and samples, nationally and internationally, would facilitate robust multicentre studies and avoid duplication of effort., (© 2021 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Human African trypanosomiasis (HAT) is a neglected tropical disease, with a population of 70 million at risk. Current treatment options are limited. In the search for new therapeutics, the repurposing of the broad-spectrum antiprotozoal drug fexinidazole has completed Phase III trials with the anticipation that it will be the first oral treatment for HAT. This study used the recently validated bioluminescence imaging model to assess the dose and rate of kill effect of fexinidazole in infected mice, and the dose-dependent effect of fexinidazole on trypanosome infection. Pharmacokinetics of fexinidazole in plasma and central nervous system (CNS) compartments were similar in both infected and uninfected mice. Drug distribution within the CNS was further examined by microdialysis, showing similar levels in the cortex and hippocampus. However, high variability in drug distribution and exposure was found between mice., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The causative agent of tuberculosis, Mycobacterium tuberculosis (M. tuberculosis), contains an abundant cell wall glycolipid and a crucial virulence factor, trehalose-6,6'-dimycolate (TDM). TDM causes delay of phagosome maturation and thus promotes survival of mycobacteria inside host macrophages by a not fully understood mechanism. TDM signals through the Monocyte-INducible C-type LEctin (Mincle), a recently identified pattern recognition receptor. Here we show that recruitment of Mincle by TDM coupled to immunoglobulin (Ig)G-opsonised beads during Fcγ receptor (FcγR)-mediated phagocytosis interferes with phagosome maturation. In addition, modulation of phagosome maturation by TDM requires SH2-domain-containing inositol polyphosphate 5' phosphatase (SHP-1) and the FcγRIIB, which strongly suggests inhibitory downstream signalling of Mincle during phagosome formation. Overall, our study reveals important mechanisms contributing to the virulence of TDM.

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites. Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection. Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

Objectives: To optimize the Trypanosoma brucei brucei GVR35 VSL-2 bioluminescent strain as an innovative drug evaluation model for late-stage human African trypanosomiasis., Methods: An IVIS® Lumina II imaging system was used to detect bioluminescent T. b. brucei GVR35 parasites in mice to evaluate parasite localization and disease progression. Drug treatment was assessed using qualitative bioluminescence imaging and real-time quantitative PCR (qPCR)., Results: We have shown that drug dose-response can be evaluated using bioluminescence imaging and confirmed quantification of tissue parasite load using qPCR. The model was also able to detect drug relapse earlier than the traditional blood film detection and even in the absence of any detectable peripheral parasites., Conclusions: We have developed and optimized a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo and reduce the current 180 day drug relapse experiment to a 90 day model. The non-invasive in vivo imaging model reduces the time required to assess preclinical efficacy of new anti-trypanosomal drugs., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. Like those of other members of the family, BTV virions are nonenveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and in the delivery of the transcriptionally active core to the cell cytoplasm. Although the importance of the endocytic pathway in BTV entry has been reported, detailed analyses of entry and the role of each protein in virus trafficking have not been possible due to the lack of availability of a tagged virus. Here, for the first time, we report on the successful manipulation of a segmented genome of a nonenveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed the separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of the trafficking of BTV farther downstream in different host cells. In addition, the tagging technology has potential for transferable application to other nonenveloped complex viruses., Importance: Live-virus trafficking in host cells has been highly informative on the interactions between virus and host cells. Although the insertion of fluorescent markers into viral genomes has made it possible to study the trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have imposed practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of bluetongue virus (BTV), a complex nonenveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies of both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids., Methodology/principal Findings: Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5'-variant surface glycoprotein untranslated region (UTR) and a downstream 3'-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter., Conclusions/significance: The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.

Noninvasive in vivo fluorescence imaging of small animals as a method in preclinical research has developed considerably in recent years, and is used widely across a variety of disciplines such as oncology and infectious disease research. It provides a means of detecting a fluorescent signal within a living animal reflecting specific, mostly disease-related, processes, such as parts of the host immune response, inflammation, cancer growth or presence of pathogens. As well as offering many advantages as a standalone technique, it can also be highly complementary to other imaging modalities. This review discusses aspects of light distribution in animal tissue and the implications on in vivo imaging; the most widely used imaging techniques including planar and tomographic imaging; advantages and challenges of the techniques; fluorescent contrast agents and some examples of applications. Rather than in detail reviewing studies using in vivo fluorescence imaging, we focus on the principles and practicalities of the method itself, so that the reader can apply these to their own research question., (© 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.)

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

The purpose of this article is to reinforce the need for all dental clinicians to perform a complete lymph node examination on every patient, regardless of age, gender, or chief complaint. As early diagnosis provides for the best prognosis, head and neck lymph node palpation may be the earliest indicator of infection or neoplasia. This article provides the rationale for lymph node examination, the palpation techniques for the clinician to utilize, and the anatomic locations and descriptions of lymph nodes.

The pathogenic Escherichia coli strain E. coli K1 is a primary causative agent of neonatal meningitis. Understanding how these bacteria cross the blood-brain barrier is vital to develop therapeutics. Here, we describe the use of live-cell imaging techniques to study E. coli K1 interactions with cellular markers following infection of human brain microvascular endothelial cells, a model system of the blood-brain barrier. We also discuss optimization of endothelial cell transfection conditions using nonviral transfection technique, bacterial labeling techniques, and in vitro assays to screen for fluorescent bacteria that retain their ability to invade host cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: This trial describes a first-in-man evaluation of RH1, a novel bioreductive drug activated by DT-diaphorase (DTD), an enzyme overexpressed in many tumours., Patients and Methods: A dose-escalation phase I trial of RH1 was carried out. The primary objective was to establish the maximum tolerated dose (MTD) of RH1. Secondary objectives were assessment of toxicity, pharmacokinetic determination of RH1 and pharmacodynamic assessment of drug effect through measurement of DNA cross linking in peripheral blood mononuclear cells (PBMCs) and tumour, DTD activity in tumour and NAD(P)H:quinone oxidoreductase 1 (NQO1) polymorphism status., Results: Eighteen patients of World Health Organization performance status of zero to one with advanced refractory solid malignancies were enrolled. MTD was 1430 μg/m(2)/day with reversible bone marrow suppression being dose limiting. Plasma pharmacokinetic analysis showed RH1 is rapidly cleared from blood (t(1/2) = 12.3 min), with AUC increasing proportionately with dose. The comet-X assay demonstrated dose-related increases in DNA cross linking in PBMCs. DNA cross linking was demonstrated in tumours, even with low levels of DTD. Only one patient was homozygous for NQO1 polymorphism precluding any conclusion of its effect., Conclusions: RH1 was well tolerated with predictable and manageable toxicity. The MTD of 1430 μg/m(2)/day is the dose recommended for phase II trials. The biomarkers of DNA cross linking, DTD activity and NQO1 status have been validated and clinically developed.

Purpose: Lung cancer is the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) lacks validated biomarkers to predict treatment response. This study investigated whether circulating tumor cells (CTCs) are detectable in patients with NSCLC and what their ability might be to provide prognostic information and/or early indication of patient response to conventional therapy., Patients and Methods: In this single-center prospective study, blood samples for CTC analysis were obtained from 101 patients with previously untreated, stage III or IV NSCLC both before and after administration of one cycle of standard chemotherapy. CTCs were measured using a semiautomated, epithelial cell adhesion molecule-based immunomagnetic technique., Results: The number of CTCs in 7.5 mL of blood was higher in patients with stage IV NSCLC (n = 60; range, 0 to 146) compared with patients with stage IIIB (n = 27; range, 0 to 3) or IIIA disease (n = 14; no CTCs detected). In univariate analysis, progression-free survival was 6.8 v 2.4 months with P < .001, and overall survival (OS) was 8.1 v 4.3 months with P < .001 for patients with fewer than five CTCs compared with five or more CTCs before chemotherapy, respectively. In multivariate analysis, CTC number was the strongest predictor of OS (hazard ratio [HR], 7.92; 95% CI, 2.85 to 22.01; P < .001), and the point estimate of the HR was increased with incorporation of a second CTC sample that was taken after one cycle of chemotherapy (HR, 15.65; 95% CI, 3.63 to 67.53; P < .001)., Conclusion: CTCs are detectable in patients with stage IV NSCLC and are a novel prognostic factor for this disease. Further validation is warranted before routine clinical application.

Recent advances in technology now permit robust and reproducible detection of circulating tumour cells (CTCs) from a simple blood test. Standardization in methodology has been instrumental in facilitating multicentre trials with the purpose of evaluating the clinical utility of CTCs. We review the current body of evidence supporting the prognostic value of CTC enumeration in breast, prostate and colorectal cancer, using standardized approaches, and studies evaluating the correlation of CTC number with radiological outcome. The exploitation of CTCs in cancer management, however, is now extending beyond prognostication. As technologies emerge to characterize CTCs at the molecular level, biological information can be obtained in real time, with the promise of serving as a 'surrogate tumour biopsy'. Current studies illuminate the potential of CTCs as pharmacodynamic and predictive biomarkers and potentially their use in revealing drug resistance in real time. Approaches for CTC characterization are summarized and the potential of CTCs in cancer patient management exemplified via the detection of epidermal growth factor receptor mutations from CTCs in patients with non-small cell lung cancer. The opportunity to learn more about the biology of metastasis through CTC analysis is now being realized with the horizon of CTC-guided development of novel anticancer therapies.

The introduction of new anticancer drugs into the clinic is often hampered by a lack of qualified biomarkers. Method validation is indispensable to successful biomarker qualification and is also a regulatory requirement. Recently, the fit-for-purpose approach has been developed to promote flexible yet rigorous biomarker method validation, although its full implications are often overlooked. This review aims to clarify many of the scientific and regulatory issues surrounding biomarker method validation and the analysis of samples collected from clinical trial subjects. It also strives to provide clear guidance on validation strategies for each of the five categories that define the majority of biomarker assays, citing specific examples., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Rituximab is a chimeric anti-CD20 monoclonal antibody that has revolutionised the treatment of many B-cell malignancies, and is now increasingly being used in non-malignant conditions such as auto-immune disorders. Serum rituximab levels are highly variable in patients receiving similar 'standard' approved doses. Little is known regarding the factors that affect serum rituximab concentration and that in turn may influence clinical outcome. In order to provide a tool that may ultimately enable patient specific dosing of rituximab therapy, we have validated a reliable, robust ELISA for the quantitation of serum rituximab levels to provide accurate pharmacokinetic (PK) data that will guide the optimisation of rituximab dosing regimes. Extensive validation of the assay was performed in order to utilise the assay for clinical applications. The within and between day plate coating reproducibility was tested and proved a robust starting platform for the assay. The within day precision for the assay was determined using spiked serum samples and was shown to have a coefficient of variation (CV) of <10% with an accuracy between 91 and 125%. The between day precision (CV) was <25% with an accuracy between 95 and 109%. Dilution linearity and parallelism were demonstrated. Spike recovery for all concentrations and donors was shown to be within +/-15% on average, with a CV below 10%. This assay is highly accurate and reproducible in determining the levels of rituximab in spiked serum samples. It meets stringent acceptance criteria, is fit for purpose, and is currently being applied to several clinical trials incorporating rituximab in the treatment of lymphoma. This assay represents a useful tool for clinical application of this widely used therapeutic., (2010 Elsevier B.V. All rights reserved.)

Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI., Methodology/principal Findings: One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging., Conclusions/significance: While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the P(mtbB) is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.

The differentiation of B cells into Ig-secreting plasma cells requires the expansion of secretory organelles to cope with the increased cargo load. To evaluate the timeline of this process, we have quantitated the kinetics of secretory organelle expansion relative to Ig secretion and examined regulatory components of secretory transport following in vitro activation of human B lymphocytes. Unstimulated B cells contain minimal endomembranes. After activation, ER membrane induction appears as tightly packed spherical structures of 0.5-1 mum diameter concentrated in a juxtanuclear position. When the cells differentiate into plasmablasts, there is dramatic cell-size increase, but the ER remains concentrated close to the nucleus and only later fills the entire cell. In sharp contrast, previous studies in other cell types have found that the ER expands in synchrony with increasing cell size during interphase, by extension of ER tubules under the PM. In this study, the Golgi remains consistently as a single juxtanuclear structure but linearly expands sixfold in volume during B cell activation. Furthermore, following active cell proliferation, ER exit sites proliferate rapidly, increasing almost fourfold in number, in parallel with a sharp increase in Ig secretion. These findings demonstrate that the control of organelle biogenesis and expansion in primary human B cells are differentially regulated by cargo flux caused by Ig synthesis.

Validated assays of circulating biomarkers of angiogenesis to predict and determine the efficacy of vascular-targeted anticancer drugs would facilitate successful drug development. Multiple biomarker candidates exist and a multiplex approach was sought to minimise the requisite patient blood volume and to aid selection of those biomarkers with greatest potential clinical utility. Validation of the SearchLight multiplex ELISA platform comprising two multiplex assays of nine potential angiogenesis biomarkers was conducted (plex 1; VEGF R1 and R2, IL-8, KGF, PlGF; plex 2; PDGFbb, HGF, FGFb and VEGF). The study focused on instrument qualification, analyte specificity within the multiplex format, assay precision and reproducibility. No evidence was found within the multiplex that signals output from one analyte impinged on another or that antibody cross-reactivity occurred. Spike recovery for 5 between-experiment repeats was within +/-15% of input values for 7 of the 9 multiplexed analytes, with a coefficient of variation (CV) of <20% for 6 of the 9 analytes. Plasma samples from 8 ovarian cancer patients (who were not receiving therapy) were assessed using the two multiplexes on this platform to explore the likely baseline variability in this disease context. This study suggests that the platform and the multiplex approach will be useful to evaluate pharmacodynamic responses to vascular targeted therapy in early clinical trials.

Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

Within the era of molecularly targeted anticancer agents, it has become increasingly important to provide proof of mechanism as early on as possible in the drug development cycle, especially in the clinic. Selective activation of apoptosis is often cited as one of the major goals of cancer chemotherapy. Thus, the present minireview focuses on a discussion of the pros and cons of a variety of methodological approaches to detect different components of the apoptotic cascade as potential biomarkers of programmed cell death. The bulk of the discussion centres on serological assays utilising the technique of ELISA, since here there is an obvious advantage of sampling multiple time points. Potential biomarkers of apoptosis including circulating tumour cells, cytokeratins and DNA nucleosomes are discussed at length. However, accepting that a single biomarker may not have the power to predict proof of concept and patient outcome, it is clear that in the future more emphasis will be placed on technologies that can analyse panels of biomarkers in small volumes of samples. To this end the increased throughput afforded by multiplex ELISA technologies is discussed.

M30 and M65 are ELISAs that detect different circulating forms of cytokeratin 18. Using the aurora kinase inhibitor AZD1152 and the SW620 human colon cancer xenograft, experiments were conducted to qualify preclinically both assays as serologic biomarkers of cell death. Using two different apoptotic markers, the kinetics of cell death induced by AZD1152 was first characterized in vitro in three different cell lines and shown to peak 5 to 7 days after drug addition. Treatment of non-tumor-bearing rats with AZD1152 (25 mg/kg) produced no alterations in circulating baseline values of M30 and M65 antigens. In treated, tumor-bearing animals, M30 detected a 2- to 3-fold (P < 0.05) increase in plasma antigen levels by day 5 compared with controls. This correlated to a 3-fold increase in the number of apoptotic cells detected on day 5 in SW620 xenografts using immunohistochemistry. By contrast, M65 did not detect a drug-induced increase in circulating antigen levels at day 5. However, M65 plasma levels correlated to changes in tumor growth in control animals (r(2) = 0.93; P < 0.01) and also followed the magnitude of the temporal effect of AZD1152 on tumor growth. An intermediate but active dose of AZD1152 (12.5 mg/kg) produced a less significant increase in M30 plasma levels at day 5. It was also confirmed that the plasma profiles of M30 and M65 mirrored closely those measured in whole tumor lysates. We conclude that M30 is a pharmacodynamic biomarker of AZD1152-induced apoptosis in the SW620 xenograft model, whereas M65 is a biomarker of therapeutic response.

Over recent years the role of biomarkers in anticancer drug development has expanded across a spectrum of applications ranging from research tool during early discovery to surrogate endpoint in the clinic. However, in Europe when biomarker measurements are performed on samples collected from subjects entered into clinical trials of new investigational agents, laboratories conducting these analyses become subject to the Clinical Trials Regulations. While these regulations are not specific in their requirements of research laboratories, quality assurance and in particular assay validation are essential. This review, therefore, focuses on a discussion of current thinking in biomarker assay validation. Five categories define the majority of biomarker assays from 'absolute quantitation' to 'categorical'. Validation must therefore take account of both the position of the biomarker in the spectrum towards clinical end point and the level of quantitation inherent in the methodology. Biomarker assay validation should be performed ideally in stages on 'a fit for purpose' basis avoiding unnecessarily dogmatic adherence to rigid guidelines but with careful monitoring of progress at the end of each stage. These principles are illustrated with two specific examples: (a) absolute quantitation of protein biomarkers by mass spectrometry and (b) the M30 and M65 ELISA assays as surrogate end points of cell death.

Purpose: RH1 is a novel anticancer agent with potent DNA-cross linking activity. RH1 has the potential to be activated within tumors over expressing NQO1, giving maximal antitumour activity with reduced toxicity in normal tissues. RH1 has recently completed a Cancer Research UK sponsored phase I clinical trial at two different centers in the United Kingdom. The comet-X assay was a secondary endpoint in this trial and assay validation was necessary. We describe here this validation process. Whilst it is impossible to cover all variations/conditions of a pharmacodynamic assay, we have strived to evaluate and demonstrate that this assay conforms to the three R's of validation, that is robustness, reliability and reproducibility., Methods: K562 and peripheral blood mononuclear cells were treated with either radiation alone, or with a combination of radiation and drug. These samples were then embedded in low melting point agarose and subjected to a modified version of the alkaline single cell gel electrophoresis (Comet) assay, described here as the comet-X assay. Variations in the preparation, electrophoresis, storage and scoring of these samples was investigated. In addition radiation and drug dose response curves were constructed. Finally stability of QC standards was investigated over a 30-month period., Results: We have demonstrated a linear radiation-dose response in cells up to 20 Gy and drug induced DNA cross-linking up to 50 nM. From the radiation dose response curves we were able to show that the relative inaccuracy measured against a global mean value was less than 25% and the relative (within day) imprecision was less than 30% over all doses. Between day runs produced an intra assay imprecision of 21.2%. Variables involved in the electrophoresis process showed the voltage across all slides in the tank ranged from 3.1 to -2.0 (mV) whilst the current ranged from 0.8-5.5 mA. QC standards were prepared from PBMCs of healthy donors and frozen at -80 degrees C. The stability of these frozen QC standards was measured over a 30-month period. No significant deterioration in any of the control, irradiated or drug treated samples was observed., Conclusions: The comet-X assay has been shown to be a robust, reliable and reproducible assay. It is ideally suited for the evaluation of the pharmacodynamic effects of DNA cross-linking agents undergoing early clinical trials. Furthermore, this assay may provide valuable data, in conjunction with pharmacokinetics, when measuring toxicity and efficacy as part of the RH1 phase I clinical trial.

Transport through the secretory pathway begins with COPII regulation of ER export. Driven by the Sar1 GTPase cycle, cytosolic COPII proteins exchange on and off the membrane at specific sites on the ER to regulate cargo exit. Here recent developments in COPII research are discussed, particularly the use of live-cell imaging, which has revealed surprising insights into the coat's role. The seemingly static ER exit sites are in fact highly dynamic, and the ability to visualise trafficking processes in intact living cells has highlighted the adaptable nature of COPII in cargo transport and the emerging roles of auxiliary factors.

The use of green fluorescent protein (GFP) chimeras to illuminate the secretory pathway in living cells has provided a wealth of information on the mechanisms of protein retention, sorting, and recycling. A wide variety of microscopic techniques, including time-lapse imaging, double-labeling, quantitation, photobleaching, and energy transfer approaches, have been utilized to explore the organization of the early secretory pathway. In this chapter we focus on the application of GFP technology to gain insight into the dynamics of ERGIC-53, a putative cargo receptor localized to the early secretory pathway, and the way in which photobleaching approaches have provided insight into its transport.

A high level of hypoxia in solid tumours is an adverse prognostic factor for the poor outcome of cancer patients following treatment. This review describes the status of research into finding a practical method for measuring hypoxia and treating hypoxic tumours. The application of such methodology would enable the selection of head and neck cancer treatment based on an individual's tumour oxygenation status. This individualization would include the selection not only of surgery or radiotherapy, but also of novel hypoxia-modification strategies.

Background: Radiotherapy for the control of cancer, either alone or in conjunction with chemotherapy, is often limited by normal tissue toxicity including haematopoietic toxicity. Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species that are associated with radiation-induced cytotoxicity. The antioxidant enzyme manganese superoxide dismutase (SOD2) catalyzes the dismutation of the superoxide anions into hydrogen peroxide., Methods: We have investigated the potential of SOD2 overexpression, through retroviral gene transfer using a retrovirus optimized for transcription in early haematopoietic cells, to enhance the radioresistance of a human erythroleukaemic cell line and primary murine bone marrow. Using these as in vitro models we have investigated whether SOD2 gene therapy may be suitable for the protection of the haematopoietic compartment from the effects of ionizing radiation., Results: Here we demonstrate using both biological and physical assays that overexpression of SOD2 protects haematopoietic cells from ionizing radiation injury. Our results show that an increase in the levels of SOD2 enzymatic activity within K562 cells (from 160.7 +/- 23.6 to 321.8 +/- 45.2 U/mg protein) or primary murine haematopoietic progenitor cells leads to both a significant decrease in DNA fragmentation and a significant increase in clonogenic survival, as evident by a significant increase in Dbar (from 2.66 to 3.42Gy), SF2 (from 0.52 to 0.73) values, and a significant decrease in the alpha value (from 0.3040 +/- 0.037 to 0.0630 +/- 0.037 Gy(-1)) when compared either to cells transduced with a retroviral vector encoding eGFP alone or to the parental line., Conclusions: The results presented suggest that retroviral radioprotective gene therapy may be applicable to the haematopoietic compartment, enabling radiation dose escalation in cancer therapy., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

The tyrosine kinase inhibitor imatinib (STI 571; glivec) is a potent inhibitor of bcr-abl, c-kit and platelet-derived growth factor receptors. Imatinib was evaluated both alone and in combination with established chemotherapeutic agents in adenoid cystic carcinoma (ACC) primary cultures and established cell lines representing squamous cell carcinoma of the head and neck (HNSCC). Over 90% of ACC tumors are c-kit-positive, and these primary cultures proved to be of short-term usefulness in assessing chemosensitivity. Interaction was determined over a wide range of drug combinations using a statistical three-dimensional analysis model. Both ACC short-term cultures and HNSCC cell lines were demonstrated to have a response ranging from additive to synergistic when imatinib and cisplatin were combined. The interaction of imatinib on cisplatin-induced DNA cross-linking was further investigated using the comet-X assay. In contrast, significant antagonism was observed when imatinib and gemcitabine were combined. Since gemcitabine is activated by deoxycytidine kinase (dCK), the effect of imatinib on this enzyme was investigated. A dose-dependent inhibition of dCK was observed, highlighting this kinase as a possible additional secondary molecular target for imatinib. This work demonstrates a synergistic interaction between cisplatin and imatinib, which may prove to be clinically relevant in the future management of both ACC and HNSCC.

Purpose: The purpose of our study was to investigate the cellular accumulation, DNA cross-linking ability, and cellular toxicity of RH1 (2,5-diaziridinyl-3-[hydroxymethyl[-6-methyl-1,4-benzoquinone), a novel DNA alkylating agent currently in clinical trials. In addition, the in vivo efficacy of RH1 formulated in different vehicles was also compared., Experimental Design: RH1 is activated by the two-electron reducing enzyme NQO1 [NADPH:quinone oxidoreductase] forming a potent cytotoxic agent that cross-links DNA. We have used whole blood, cell lines, and primary explanted tumor cultures to measure both the cellular accumulation, DNA cross-linking, and cytotoxicity of RH1. Furthermore, the pharmacokinetic and pharmacodynamic characteristics of RH1 formulated in different vehicles were measured in vivo using the validated comet-X assay in mice bearing human tumor xenografts., Results: Accumulation of RH1 was shown to be both time and concentration dependent, reaching a maximum after 2 hours and correlated well with DNA cross-linking measurements. DNA cross-linking in vitro could be detected at low (1-10 nmol/L) concentrations after as little as 2 hours exposure. In primary tumor cultures, RH1 induces much higher levels of DNA cross-links at lower doses than either mitomycin C or cisplatin. In vivo efficacy testing using polyvinyl pyrrolidone, saline, or cyclodextrin as vehicles showed DNA cross-links readily detectable in all tissues examined and was enhanced when given in cyclodextrin compared with polyvinyl pyrrolidone or saline., Conclusions: RH1 represents a potent bioreductive anticancer drug, which may prove effective in the treatment of cancers, particularly those that overexpress NQO1. DNA cross-linking can be reliably measured in tissue using the validated comet-X assay.

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST-XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at -80 degrees C for at least 60 days. M30-Apoptosense plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at -80 degrees C, while at 37 degrees C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.

It is an exciting time for cancer researchers in the field of apoptotic cell death. The avalanche of discoveries over the past decade or so regarding how apoptosis is regulated begins to be exploited for therapeutic benefit as the first apoptosis-targeted drugs enter early clinical trials. This chapter provides a selective review on the development of such drugs. We also outline issues regarding the regulation and design of early clinical trials of this type of molecularly targeted agent. Finally, we discuss the biomarkers and surrogate pharmacodynamic endpoint assays currently available to chart the efficacy of apoptosis-inducing anticancer therapy.

DT-diaphorase (DTD) is an obligate two-electron reductase which bioactivates chemotherapeutic quinones. DTD levels are elevated in a number of tumour types, including non-small cell lung carcinoma, colorectal carcinoma, liver cancers and breast carcinomas, when compared to the surrounding normal tissue. The differential in DTD between tumour and normal tissue should allow targeted activation of chemotherapeutic quinones in the tumour whilst minimising normal tissue toxicity. The prototypical bioreductive drug is Mitomycin C (MMC) which is widely used in clinical practice. However, MMC is actually a relatively poor substrate for DTD and its metabolism is pH-dependent. Other bioreductive drugs have failed because of poor solubility and inability to surpass other agents in use. RH1, a novel diaziridinylbenzoquinone, is a more efficient substrate for DTD. It has been demonstrated to have anti-tumour effects both in vitro and in vivo and demonstrates a relationship between DTD expression levels and drug response. RH1 has recently entered a phase I clinical trial in solid tumours under the auspices of Cancer Research UK. Recent work has demonstrated that DTD is present in the nucleus and is associated with both p53 and the heat shock protein, HSP-70. Furthermore, DTD is inducible by several non-toxic compounds and therefore much interest has focussed on increasing the differential in DTD levels between tumour and normal tissues.

Background and Purpose: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions., Patients and Methods: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h., Results: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions., Conclusions: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.

In less than a decade the green fluorescent protein (GFP) has become one of the most popular tools for cell biologists for the study of dynamic processes in vivo. GFP has revolutionised the scientific approach for the study of vital organelles, such as the Golgi apparatus. As Golgi proteins can be tagged with GFP, in most cases without altering their targeting and function, it is a great substitute to conventional dyes used in the past to highlight this compartment. In this review, we cover the application of GFP and its spectral derivatives in the study of Golgi dynamics in mammalian and plant cells. In particular, we focus on the technique of selective photobleaching known as fluorescence recovery after photobleaching, which has successfully shed light on essential differences in the biology of the Golgi apparatus in mammalian and plant cells.