• Fragment-based hit identification: thinking in 3D.

      Morley, A; Pugliese, A; Birchall, K; Bower, J; Brennan, P; Brown, N; Chapman, T; Drysdale, M; Gilbert, I; Hoelder, S; et al. (2013-12)
      The identification of high-quality hits during the early phases of drug discovery is essential if projects are to have a realistic chance of progressing into clinical development and delivering marketed drugs. As the pharmaceutical industry goes through unprecedented change, there are increasing opportunities to collaborate via pre-competitive networks to marshal multifunctional resources and knowledge to drive impactful, innovative science. The 3D Fragment Consortium is developing fragment-screening libraries with enhanced 3D characteristics and evaluating their effect on the quality of fragment-based hit identification (FBHI) projects.
    • Frailty in older patients undergoing emergency laparotomy: results from the UK observational Emergency Laparotomy and Frailty (ELF) study

      Parmar, Kat; Law, J; Carter, B; Hewitt, J; Boyle, JM; Casey, P; Maitra, I; Farrell, IS; Pearce, L; Moug, SJ; et al. (2019)
      OBJECTIVE: This study aimed to document the prevalence of frailty in older adults undergoing emergency laparotomy and to explore relationships between frailty and postoperative morbidity and mortality. SUMMARY BACKGROUND DATA: The majority of adults undergoing emergency laparotomy are older adults (³65 y) that carry the highest mortality. Improved understanding is urgently needed to allow development of targeted interventions. METHODS: An observational multicenter (n=49) UK study was performed (March-June 2017). All older adults undergoing emergency laparotomy were included. Preoperative frailty score was calculated using the progressive Clinical Frailty Score (CFS): 1 (very fit) to 7 (severely frail). Primary outcome measures were the prevalence of frailty (CFS 5-7) and its association to mortality at 90 days postoperative. Secondary outcomes included 30-day mortality and morbidity, length of critical care, and overall hospital stay. RESULTS: A total of 937 older adults underwent emergency laparotomy: frailty was present in 20%. Ninety-day mortality was 19.5%. After age and sex adjustment, the risk of 90-day mortality was directly associated with frailty: CFS 5 adjusted odds ratio (aOR) 3.18 [95% confidence interval (CI), 1.24-8.14] and CFS 6/7 aOR 6á10 (95% CI, 2.26-16.45) compared with CFS 1. Similar associations were found for 30-day mortality. Increasing frailty was also associated with increased risk of complications, length of Intensive Care Unit, and overall hospital stay. CONCLUSIONS: A fifth of older adults undergoing emergency laparotomy are frail. The presence of frailty is associated with greater risks of postoperative mortality and morbidity and is independent of age. Frailty scoring should be integrated into acute surgical assessment practice to aid decision-making and development of novel postoperative strategies.
    • A framework for advancing our understanding of cancer-associated fibroblasts

      Sahai, E; Astsaturov, I; Cukierman, E; DeNardo, DG; Egeblad, M; Evans, RM; Fearon, D; Greten, FR; Hingorani, SR; Hunter, T; et al. (2020)
      Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.
    • Free radical formation from anthracycline antitumour agents and model systems--I. Model naphthoquinones and anthraquinones.

      Dodd, Nicholas J F; Mukherjee, Tulsi; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Wilmslow Road, Withington, Manchester M20 9BX, UK (1984-02-01)
      Several naphthoquinones and anthraquinones were chosen as simple models of the anthracycline drugs and their semiquinone radical anions were generated by various methods. With the exception of 1,4-naphthoquinone, all of the quinones studied gave radicals that were highly reactive with oxygen, but which, in its absence, were stable over a limited pH range. The radicals were studied using electron spin resonance (ESR) spectroscopy and an examination was made of the effect on the distribution of the unpaired electron, of introducing various groups into the conjugated ring system. Hydroxyl groups capable of participating in strong intramolecular hydrogen bonding with neighbouring carbonyl groups had a marked influence on electron distribution and reduced the effects of intermolecular hydrogen bonding of the radicals with solvent molecules.
    • Free radical production from normal and adriamycin-treated rat cardiac sarcosomes

      Thornalley, Paul J; Department of Biophysical Chemistry, Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester M20 9BX, U.K. (1985)
    • Free radical reactions in the coenzyme B12 system.

      Blackburn, R; Kyaw, M; Phillips, G O; Swallow, A John (1975)
    • Free radical scavenging properties of melanin interaction of eu- and pheo-melanin models with reducing and oxidising radicals.

      Rózanowska, Malgorzata; Sarna, Tadeusz; Land, Edward J; Truscott, T G; Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, Kraków, Poland. (1999-03)
      The human skin and eye melanin is commonly viewed as an efficient photoprotective agent. To elucidate the molecular mechanism of the melanin-dependent photoprotection, we studied the interaction of two synthetic melanins, dopa-melanin and cysteinyldopa-melanin, with a wide range of oxidising and reducing free radicals using the pulse radiolysis technique. We have found that although both types of free radicals could efficiently interact with the synthetic melanins, their radical scavenging properties depended, in a complex way, on the redox potential, the electric charge and the lifetime of the radicals. Repetitive pulsing experiments, in which the free radicals, probing the polymer redox sites, were generated from four different viologens, indicated that the eumelanin model had more reduced than oxidised groups accessible to reaction with the radicals. Although with many radicals studied, melanin interacted via simple one-electron transfer processes, the reaction of both melanins with the strongly oxidising peroxyl radical from carbon tetrachloride, involved radical addition. Our study suggests that the free radical scavenging properties of melanin may be important in the protection of melanotic cells against free radical damage, particularly if the reactive radicals are generated in close proximity to the pigment granules.
    • Free radicals and food irradiation.

      Dodd, Nicholas J F; CRC Department of Biophysics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, U.K. (1995)
      Ionizing radiation can be used to control insect and microbial infestation of foodstuffs, inhibit sprouting, delay ripening and reduce the dangers from food-poisoning bacteria. Irradiation produces free radicals, most of which decay rapidly, although some are more persistent. These latter radicals can be detected and characterized by electron spin resonance (ESR). In bone and other calcified tissues, the radiation-induced radicals are distinguishable from naturally occurring radicals, and their stability makes them ideal for radiation dosimetry. The radicals induced in plant material, such as seeds and dried spices, are generally indistinguishable from the endogenous radicals and decay over a period of days or weeks. However, in many of these materials, a radiation-specific radical can be detected at low concentration, thereby permitting identification of irradiated samples, although precluding accurate dosimetry. ESR, although not universally applicable, currently provides the most specific method for the detection of irradiated food.
    • Frequency of down-regulation of individual HLA-A and -B alleles in cervical carcinomas in relation to TAP-1 expression.

      Keating, P J; Cromme, F V; Duggan-Keen, Margaret F; Snijders, P J; Walboomers, Jan M; Hunter, Robin D; Dyer, P A; Stern, Peter L; Cancer Research Campaign Department of Immunology, Paterson Institute for Cancer Research, Manchester, UK. (1995-08)
      The development of cervical carcinoma is strongly associated with specific types of human papillomaviruses (HPVs). A role for cellular immunity in cervical disease is supported by the increased occurrence of HPV-associated lesions in immunosuppressed individuals. Upon viral infection or malignant transformation, ensuing alterations in gene expression result in the generation of novel sets of peptides which can form complexes with specific HLA class I heavy chains and beta 2-microglobulin. These are then expressed at the cell surface as potential targets for specific T cells. In this study of 100 carcinomas HLA-A and -B class I expression by the tumour cells was down-regulated at one or more alleles in at least 73% of cervical carcinomas. Interference with the transporter associated with antigen presentation (TAP), which translocates cytosolic peptides from endogenously synthesised proteins (e.g. viral) into the lumen of the endoplasmic reticulum was found in 38% of the HLA class I down-regulated tumours. Loss of expression for common HLA class I alleles ranged from 36% to 71%, and such changes might be expected to influence specific immunogenic peptide presentation and consequent immune recognition. These results underline the importance of single as well as multiple allelic loss in cervical neoplasia and have important implications for attempts to intervene immunologically in cervical cancer.
    • Frequency of fibroblast growth factor receptor 3 mutations in sporadic tumours.

      Sibley, Kathryn; Stern, Peter L; Knowles, Margaret A; ICRF Clinical Centre, St. James's University Hospital, Leeds, LS9 7TF, UK. (2001-07-19)
      Mutations in FGFR3 have been identified in several tumour types including bladder carcinoma, cervical carcinoma, and multiple myeloma. In bladder carcinoma, we recently identified FGFR3 mutations in 41% of tumours, making this the most frequently mutated putative oncogene identified in bladder cancer to date. We have now investigated the frequency of FGFR3 mutation in a panel of 125 tumours and 13 cell lines from various other organs. We analysed the mutation hotspots in exons 7, 10 and 15 by direct DNA sequencing, and found one mutation in exon 7 (S249C) in 1/28 (3.5%) cervical tumours. Mutations were not detected in stomach, rectum, colon, prostate, ovarian, breast, brain, or renal tumours, nor were they found in any of the cell lines included in this study. We conclude that FGFR3 is commonly mutated in bladder carcinoma and only rarely in cervical carcinoma. Several tumour types appear not to possess any mutations in FGFR3, suggesting that these mutations are important only in the development of certain types of tumour.
    • Frequency of human T regulatory cells in peripheral blood is significantly reduced by cryopreservation.

      Elkord, Eyad; Clinical Immunotherapy Laboratory, Department of Medical Oncology, University of Manchester, Wilmslow Road, Manchester M204BX, UK. eelkord@picr.man.ac.uk (2009-08-15)
      Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation.
    • Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester.

      Jackson, Peta E; Hall, C N; Badawi, Alaa F; O'Connor, Peter J; Cooper, Donald P; Povey, Andrew C; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, United Kingdom. (1996-05)
      Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O6-methyldeoxyguanosine (O6-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O6-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O6-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 mumol O6-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O6-MedG in the DNA from both their tumor and normal tissue. Ki-ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC-->AT transitions: 28% and 8% were GC-->TA and CG-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC-->TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O6-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC-->AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype.
    • The frequency of osteolytic bone metastasis is determined by conditions of the soil, not the number of seeds; evidence from in vivo models of breast and prostate cancer.

      Wang, N; Reeves, Kimberley J; Brown, H; Fowles, A; Docherty, F; Ottewell, P; Croucher, P; Holen, I; Eaton, C; The Mellanby Centre for Bone Research, Department of Human Metabolism, Medical School, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX (2015)
      While both preclinical and clinical studies suggest that the frequency of growing skeletal metastases is elevated in individuals with higher bone turnover, it is unclear whether this is a result of increased numbers of tumour cells arriving in active sites or of higher numbers of tumour cells being induced to divide by the bone micro-environment. Here we have investigated how the differences in bone turnover affect seeding of tumour cells and/or development of overt osteolytic bone metastasis using in vivo models of hormone-independent breast and prostate cancer.
    • Frequency of regulatory T cells in renal cell carcinoma patients and investigation of correlation with survival.

      Griffiths, Richard W; Elkord, Eyad; Gilham, David E; Ramani, Vijay A C; Clarke, Noel W; Stern, Peter L; Hawkins, Robert E; Department of Medical Oncology, Christie Research Centre, Paterson Institute for Cancer Research, Manchester, UK. (2007-11)
      BACKGROUND: Regulatory T cells are important in maintaining immune homeostasis, mediating peripheral tolerance and preventing autoimmunity. Increased frequencies of CD4(+)CD25(high )T regulatory (T(Reg)) cells have been documented in the peripheral blood of patients with several types of cancer consistent with a role in tumour escape from immunological control. We have investigated the presence of T(Reg) cells systemically and in situ in previously untreated patients with renal cell carcinoma (RCC). RESULTS: We have shown that there is a significant increased frequency of CD4(+)CD25(high) T cells in RCC patients (n = 49) compared to normal donors (n = 38), respectively, 2.47% versus 1.50%; P < 0.0001. We confirmed these data using the FOXP3 marker of T(Reg) cells in a subset of these patients and normal donors. The population of T(Reg) cells identified showed the expected phenotype with CD4(+)CD25(high) population in both RCC patients and normal donors contained higher proportions of CD45RO and GITR than CD4(+)CD25(-/low) populations and exhibiting suppressive activity in an anti-CD3 and anti-CD28 induced proliferation assay. CD4(+)FOXP3(+) T cells were detected in the tumour microenvironment by immunofluorescence and the numbers enumerated in lymphocytes recovered following enzymatic disaggregations of biopsies; their frequency was higher in the tumour than the peripheral blood of the same patients. The early follow up data show an association between higher peripheral blood regulatory T-cell count and adverse overall survival. CONCLUSION: These data confirm the increase of T(Reg) cells in RCC patients and provide impetus to further investigate modulation of T(Reg) activity in RCC patients as part of therapy.
    • Frequent alterations of cell cycle regulators in early-stage breast lesions as detected by immunohistochemistry.

      Marsh, K L; Varley, Jennifer; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1998-05)
      Progression through G1 phase of the eukaryotic cell cycle is tightly controlled by cyclin-dependent kinases (CDK). These proteins form part of a regulatory pathway including the cyclin-dependent kinase inhibitor (CKI) p16, D-type cyclins and the product of the retinoblastoma gene pRb. Aberration of any one of these components may lead to uncontrolled proliferation contributing to neoplasia. Three of these proteins, cyclin D1, pRb and p16, were analysed by immunohistochemistry on archival paraffin sections to determine whether expression patterns were different in preinvasive ductal carcinoma in situ (DCIS) and invasive breast tumours relative to normal. Genetic analysis of the gene encoding cyclin D1 (CCND1) was also carried out, using an intragenic restriction fragment-length polymorphism (RFLP) to assess possible allelic imbalance. A majority of the tumours studied (approximately 90%) showed abnormalities in expression of at least one of these proteins. Overexpression of cyclin D1 was found in approximately 49% cases, reduced expression of p16 in approximately 46% and reduced expression of pRb in approximately 37%. Allelic imbalance of cyclin D1 was found in approximately 57% cases.
    • Frequent alterations of chromosome 1 in ductal carcinoma in situ of the breast.

      Munn, K E; Walker, R A; Varley, Jennifer; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1995-04-20)
      Ductal carcinoma in situ (DCIS) of the breast is commonly described as a premalignant lesion. Using PCR to amplify DNA from areas of tumour cells which have been microdissected from fixed material, we have studied the involvement of chromosome 1 in 19 cases of DCIS. A series of microsatellite repeat polymorphisms has been used to define regions of allelic imbalance and this has confirmed the involvement in DCIS of six of the regions previously implicated in studies of invasive breast tumours. This suggests that these regions may harbour tumour suppressor genes, the inactivation of which is important for the early stages of breast tumour development. Analysis of separate ducts from within the same tumour has revealed that the same genetic alterations are not necessarily present throughout the lesion. In addition we have found that in three cases where frank invasive carcinoma is also present, similar alterations can be detected in the in situ and invasive component.
    • Frequent loss of function mutations in TGF beta R1 and TGF beta R2 identify hair follicle bulge stem cells as the cell of origin for cutaneous squamous cell carcinoma

      Cammareri, P; Rose, A; Vincent, D; Libertini, S; Ridgway, R; Athineos, D; Coates, P; McHugh, A; Pourreyron, C; Larsson, J; et al. (2015)
    • Frequent loss of function mutations in TGF beta R1 and TGF beta R2 implicate hair follicle bulge stem cells as a cell of origin of cutaneous squamous cell carcinoma.

      Cammareri, P; Rose, A; Vincent, D; Wang, J; Nagano, A; Libertini, S; Ridgway, R; McHugh, A; Pourreyron, C; Spender, L; et al. (2016)
    • Friend disease in vitro.

      Dexter, T Michael; Allen, Terence D; Testa, Nydia G; Scolnick, E (1981-09-01)
      In long-term marrow cultures, hemopoiesis can be maintained for several months, although erythropoiesis is normally suppressed at the most primitive level of development (the erythroid colony-forming cells). Infection of these cultures with a viral complex combining helper-independent murine leukemia virus (F-MuLV) and a spleen focus-forming virus (SFFVp) results in a productive infection of both the replication defective SFFVp and the F-MuLV. After infection, the cultures show a dramatic elevation in the numbers of late erythroid progenitor cells (CFU-E), many of which will grow in the absence of added erythropoietin, and a transient erythropoietin, independent erythropoiesis, including the production of mature, enucleated erythrocytes. Hemopoiesis eventually declines, with no evidence for the generation of Friend tumor cells. When erythropoiesis is induced in the long-term cultures by addition of anemic mouse serum before infection by polycythemia-inducing Friend virus, the generation of erythropoietin-independent CFU-E and erythrocyte formation is followed by the sustained production (greater than 40 wk) of primitive erythroid cells with low spontaneous levels (less than 5%) of hemoglobinization. Although these cells will produce spleen colonies in irradiated mice and can be cloned in soft-gel media, they do not produce autonomous, permanently growing cell lines in vitro, i.e., they retain a dependency upon the marrow-adherent layer for their continued growth. However, following a further passage on a "virgin" marrow environment, permanent cell lines can be established that are able to grow independently of environmental influences. Thus, this system is the first description of a complete in vitro system for the reproducible production and isolation of Friend virus-induced erythroid cell lines.
    • From live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label.

      Drummond, Sheona P; Allen, Terence D; Department of Structural Cell Biology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. (2008)
      The identification and characterization of many biological substructures at high resolution requires the use of electron microscopy (EM) technologies. Scanning electron microscopy (SEM) allows the resolution of cellular structures to approximately 3 nm and has facilitated the direct visualization of macromolecular structures, such as nuclear pore complexes (NPCs), which are essential for nucleo-cytoplasmic molecular trafficking. However, SEM generates only static images of fixed samples and therefore cannot give unambiguous information about protein dynamics. The investigation of active processes and analysis of protein dynamics has greatly benefited from the development of molecular biology techniques whereby vectors can be generated and transfected into tissue culture cells for the expression of specific proteins tagged with a fluorescent moiety for real-time light microscopy visualization. As light microscopy is limited in its powers of resolution relative to electron microscopy, it has been important to adapt a protocol for the processing of samples for real-time imaging by conventional light microscopy with protein labels that can also be identified by SEM. This allows correlation of dynamic events with high resolution molecular and structural identification. This method describes the use of GFP for tracking the dynamic distribution of NPC components in real-time throughout the cell cycle and for high resolution immuno-SEM labeling to determine localization at the nanometer level.