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1.  Adherence to NICE guidelines for new glaucoma referrals 
Eye  2013;27(10):1230.
PMCID: PMC3806559
2.  Adherence to NICE guidelines for new glaucoma referrals 
Eye  2013;27(4):571-572.
PMCID: PMC3626014  PMID: 23429410
3.  Echocardiogram Study To Evaluate the Effect of the Novel Hepatitis C Virus NS5A Inhibitor GSK2336805 on Cardiac Contractility in Healthy Subjects 
Antimicrobial Agents and Chemotherapy  2013;57(10):5141-5143.
GSK2336805 is a hepatitis C virus NS5A inhibitor in clinical development for the treatment of chronic hepatitis C virus infection. This was a single-center, randomized, double-blind, placebo-controlled, two-period crossover study in healthy adults to evaluate the effects of a single 150-mg dose of GSK2336805 on echocardiographic measures of contractility. GSK2336805 had no effect on ejection fraction, and there was no significant correlation between GSK2336805 plasma concentration and ejection fraction. (This study has been registered at under registration no. NCT01424540.)
PMCID: PMC3811415  PMID: 23856771
4.  Safety, Tolerability, Pharmacokinetics, and Antiviral Activity of GSK2336805, an Inhibitor of Hepatitis C Virus (HCV) NS5A, in Healthy Subjects and Subjects Chronically Infected with HCV Genotype 1 
Antimicrobial Agents and Chemotherapy  2013;57(10):5037-5044.
GSK2336805 is an orally bioavailable hepatitis C virus (HCV) inhibitor working through an NS5A-mediated mechanism. This first-time-in-human study was conducted to assess the safety, tolerability, pharmacokinetics, metabolism, and efficacy of GSK2336805 in healthy subjects and subjects infected with HCV genotype 1. We performed a three-part, randomized, double-blind, placebo-controlled study in 46 healthy subjects and 23 HCV-infected subjects. After an overnight fast, healthy subjects received GSK2336805 as 10 mg, 30 mg, 30 mg plus food, and 60 mg in a single dose and 10 mg (7 days), 30 mg (7 days), and 75 mg (14 days) in a once-daily multiple dose. Subjects with HCV received GSK2336805 as a 1- to 120-mg single dose. In subjects with HCV, reductions in HCV RNA were observed within 4 h and a single dose of GSK2336805 of ≥10 mg resulted in a statistically significant ≥2-log reduction in HCV RNA compared with placebo at 24 h postdose. GSK2336805 was readily absorbed in all subjects, and the half-life (t1/2) was suitable for once-daily dosing. Administration of GSK2336805 with food had no effect on plasma GSK2336805 exposure; however, absorption was delayed, with a median tmax (time to maximum concentration of drug in serum) of 4.5 versus 2.0 h. Twenty subjects who received GSK2336805 experienced mild to moderate adverse events; none were serious. GSK2336805 was well tolerated and exhibited rapid, significant antiviral activity after a single dose in HCV-infected subjects. These results support the conduct of further studies evaluating GSK2336805 administered once daily for longer durations in combination with peginterferon, ribavirin, and other direct-acting antivirals. (This study has been registered at under registration no. NCT01277692.)
PMCID: PMC3811459  PMID: 23896477
5.  Promoting exercise after a cancer diagnosis: easier said than done 
British Journal of Cancer  2014;110(4):829-830.
PMCID: PMC3929897  PMID: 24548883
6.  Validation of the EORTC QLQ-GINET21 questionnaire for assessing quality of life of patients with gastrointestinal neuroendocrine tumours 
British Journal of Cancer  2013;108(2):301-310.
Quality of life is an important end point in clinical trials, yet there are few quality of life questionnaires for neuroendocrine tumours.
This international multicentre validation study assesses the QLQ-GINET21 Quality of Life Questionnaire in 253 patients with gastrointestinal neuroendocrine tumours. All patients were requested to complete two quality of life questionnaires – the EORTC Core Quality of Life questionnaire (QLQ-C30) and the QLQ-GINET21 – at baseline, and at 3 and 6 months post-baseline; the psychometric properties of the questionnaire were then analysed.
Analysis of QLQ-GINET21 scales confirmed appropriate aggregation of the items, except for treatment-related symptoms, where weight gain showed low correlation with other questions in the scale; weight gain was therefore analysed as a single item. Internal consistency of scales using Cronbach's α coefficient was >0.7 for all parts of the QLQ-GINET21 at 6 months. Intraclass correlation was >0.85 for all scales. Discriminant validity was confirmed, with values <0.70 for all scales compared with each other.
Scores changed in accordance with alterations in performance status and in response to expected clinical changes after therapies. Mean scores were similar for pancreatic and other tumours.
The QLQ-GINET21 is a valid and responsive tool for assessing quality of life in the gut, pancreas and liver neuroendocrine tumours.
PMCID: PMC3566824  PMID: 23322194
neuroendocrine; carcinoid; quality of life; patient-reported outcome measure
7.  Validation of transgenic models of breast cancer: ductal carcinoma in situ (DCIS) and Brca1-mutation-related breast cancer 
Breast cancer online : BCO  2005;8(8):e42.
Available mouse models of ductal carcinoma in situ (DCIS) and BRCA1-mutation-related breast cancer are reviewed. The best validated mouse models of human DCIS are the conditional estrogen receptor α in mammary tissue (CERM) model initiated by deregulated estrogen receptor α and the serial explant mouse model initiated by p53 deficiency. At present the most useful and best validated mouse model of BRCA1-mutation-related breast cancer uses the cre-lox system to make a conditional Brca1 deletion targeted to mammary epithelial cells. The major shortcoming of the non-conditional Brca1 models is the high incidence of non-mammary tumor development. The use of mammary gland transplants or explants from these mice into nude hosts is one approach that could be used to circumvent this deficiency. Development and validation of a Brca1-mutation-related mouse model of basal cell breast cancer is an important next step.
PMCID: PMC3872125  PMID: 24371431
Brca1; Breast cancer; DCIS; ERα; Mouse models
8.  Effect of Feeding Fescue Seed Containing Ergot Alkaloid Toxins on Stallion Spermatogenesis and Sperm Cells 
The cellular effects of tall fescue grass–associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel-free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 h at 5 °C was to make the sperm cells less likely to undergo a calcium ionophore–induced acrosome reaction. The toxic ergot alkaloids had no effect on the freezability of sperm cells. However, if yearling colts were fed toxic ergot alkaloids, then the cytological analysis of meiotic chromosome synapsis revealed a significant increase in the proportion of pachytene spermatocytes showing unpaired sex chromosomes compared to control spermatocytes. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts.
PMCID: PMC3475766  PMID: 22524585
To gain an understanding of the genetic structure and dispersal dynamics of T. infestans populations, we analyzed the multilocus genotype of 10 microsatellite loci for 352 T. infestans collected in 21 houses of 11 rural communities in October 2002. Genetic structure was analyzed at the community and house compound levels. Analysis revealed that vector control actions affected the genetic structure of T. infestans populations. Bug populations from communities under sustained vector control (core area) were highly structured and genetic differentiation between neighboring house compounds was significant. In contrast, bug populations from communities with sporadic vector control actions were more homogeneous and lacked defined genetic clusters. Genetic differentiation between population pairs did not fit a model of isolation by distance at the microgeographical level. Evidence consistent with flight or walking bug dispersal was detected within and among communities, dispersal was more female-biased in the core area and results suggested that houses received immigrants from more than one source. Putative sources and mechanisms of re-infestation are described. These data may be use to design improved vector control strategies
PMCID: PMC3680132  PMID: 18773972
Chagas disease; Triatoma infestans; vector control; population structure; microsatellite loci
10.  Mass Spectrometry Data Collection in Parallel at Multiple Core Facilities Operating TripleTOF 5600 and Orbitrap Elite/Velos Pro/Q Exactive Mass Spectrometers 
Proteomic research can benefit from simultaneous access to multiple cutting-edge mass spectrometers. 18 core facilities responded to our investigators seeking service through the ABRF Discussion Forum. Five of the facilities selected completed four plasma proteomics experiments as routine fee-for-service. Each biological experiment entailed an iTRAQ 4-plex proteome comparison of immunodepleted plasma provided as 30 labeled-peptide fractions. Identical samples were analyzed by two AB SCIEX TripleTOF 5600 and three Thermo Orbitrap (Elite/Velos Pro/Q Exactive) instruments. 480 LC-MS/MS runs delivered >250 GB of data over two months.
We compare herein routine service analyses of three peptide fractions of different peptide abundance. Data files from each instrument were studied to develop optimal analysis parameters to compare with default parameters in Mascot Distiller 2.4, ProteinPilot 4.5 beta, AB Sciex MS Data Converter 1.3 beta, and Proteome Discover 1.3. Peak-picking for TripleTOFs was best by ProteinPilot 4.5 beta while Mascot Distiller and Proteome Discoverer were comparable for the Orbitraps.
We compared protein identification and quantitation in SwissProt 2012_07 database by Mascot Server 2.4.01 versus ProteinPilot. By all search methods, more proteins, up to two fold, were identified using the Q Exactive than others. Q Exactive excelled also at the number of unique significant peptide ion sequences. However, software-dependent impact on subsequent interpretation, due to peptide modifications, can be critical. These findings may have special implications for iTRAQ plasma proteomics. For the low abundance peptide ions, the slope of the dynamic range drop-off in the plasma proteome is uniquely sharp compared with cell lysates. Our study provides data for testable improvements in the operation of these mass spectrometers. More importantly, we have demonstrated a new affordable expedient workflow for investigators to perform proteomic experiments through the ABRF infrastructure. (We acknowledge John Cottrell for optimizing the peak-picking parameters for Mascot Distiller).
PMCID: PMC3635246
11.  blaNDM-1 Is a Chimera Likely Constructed in Acinetobacter baumannii 
Alignment of DNA sequences found upstream of aphA6 and all blaNDM-1 genes displays 100% identity. This identity continues 19 bp into the blaNDM-1 gene such that the first 6 amino acids of aphA6 and blaNDM-1 are the same. Furthermore, the percent GC content (GC%) of aphA6 is considerably lower than that of blaNDM-1 and the GC% within the blaNDM-1 structural gene changes dramatically after the first 19 bp. This is unequivocal evidence that blaNDM-1 is a chimera.
PMCID: PMC3346620  PMID: 22314529
12.  RNAi Screening Identifies TAK1 as a Potential Target for the Enhanced Efficacy of Topoisomerase Inhibitors 
Current Cancer Drug Targets  2011;11(8):976-986.
In an effort to develop strategies that improve the efficacy of existing anticancer agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve the activity of the topoisomerase I (Top1) poison camptothecin (CPT). Screening was conducted using a set of siRNAs corresponding to over 400 apoptosis-related genes in MDA-MB-231 breast cancer cells. During the course of these studies, we identified the silencing of MAP3K7 as a significant enhancer of CPT activity. Follow-up analysis of caspase activity and caspase-dependent phosphorylation of histone H2AX demonstrated that the silencing of MAP3K7 enhanced CPT-associated apoptosis. Silencing MAP3K7 also sensitized cells to additional compounds, including CPT clinical analogs. This activity was not restricted to MDA-MB-231 cells, as the silencing of MAP3K7 also sensitized the breast cancer cell line MDA-MB-468 and HCT-116 colon cancer cells. However, MAP3K7 silencing did not affect compound activity in the comparatively normal mammary epithelial cell line MCF10A, as well as some additional tumorigenic lines. MAP3K7 encodes the TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NF-κB, JNK, and p38 signaling). An analysis of TAK1 signaling pathway members revealed that the silencing of TAB2 also sensitizes MDA-MB-231 and HCT-116 cells towards CPT. These findings may offer avenues towards lowering the effective doses of Top1 inhibitors in cancer cells and, in doing so, broaden their application.
PMCID: PMC3401936  PMID: 21834757
RNAi; siRNA; Screen; Camptothecin; TAK1; MAP3K7; TAB2; TRAF6
13.  Review of Reported Clinical Information System Adverse Events in US Food and Drug Administration Databases 
Applied Clinical Informatics  2011;2(1):63-74.
The US FDA has been collecting information on medical devices involved in significant adverse advents since 1984. These reports have been used by researchers to advise clinicians on potential risks and complications of using these devices.
Research adverse events related to the use of Clinical Information Systems (CIS) as reported in FDA databases.
Three large, national, adverse event medical device databases were examined for reports pertaining to CIS.
One hundred and twenty unique reports (from over 1.4 million reports) were found, representing 32 manufacturers. The manifestations of these adverse events included: missing or incorrect data, data displayed for the wrong patient, chaos during system downtime and system unavailable for use. Analysis of these reports illustrated events associated with system design, implementation, use, and support.
The identified causes can be used by manufacturers to improve their products and by clinical facilities and providers to adjust their workflow and implementation processes appropriately. The small number of reports found indicates a need to raise awareness regarding publicly available tools for documenting problems with CIS and for additional reporting and dialog between manufacturers, organizations, and users.
PMCID: PMC3175794  PMID: 21938265
Electronic health records; information systems; mandatory reporting; medical errors; United States Food and Drug Administration
14.  A prospective study of chemotherapy-induced febrile neutropenia in the South West London Cancer Network. Interpretation of study results in light of NCAG/NCEPOD findings 
British Journal of Cancer  2010;104(3):407-412.
Chemotherapy-induced febrile neutropenia is a medical emergency complicating the treatment of many cancer patients. It is associated with considerable morbidity and mortality, as well as impacting on healthcare resources.
A prospective study of all cases of chemotherapy-induced febrile neutropenia in the South West London Cancer Network was conducted over a 4-month period. Factors including demographics, treatment history, management of febrile neutropenia and outcome were recorded.
Results and conclusi:
Our results reflect those of the recent National Chemotherapy Advisory Group (NCEPOD, 2008)/National Confidential Enquiry into Patient Outcomes and Death reports (NCAG, 2009) and highlight the need for network-wide clinical care pathways to improve outcomes in this area.
PMCID: PMC3049562  PMID: 21179036
neutropenic sepsis; chemotherapy; infection; febrile neutropenia
15.  Fit-for-purpose biomarker method validation for application in clinical trials of anticancer drugs 
British Journal of Cancer  2010;103(9):1313-1317.
Clinical development of new anticancer drugs can be compromised by a lack of qualified biomarkers. An indispensable component to successful biomarker qualification is assay validation, which is also a regulatory requirement. In order to foster flexible yet rigorous biomarker method validation, the fit-for-purpose approach has recently been developed. This minireview focuses on many of the basic issues surrounding validation of biomarker assays utilised in clinical trials. It also provides an overview on strategies to validate each of the five categories that define the majority of biomarker assays.
PMCID: PMC2990602  PMID: 20924371
biomarkers; validation; clinical trials; fit-for-purpose
16.  Obesogenic diet and physical activity behaviors: independent or associated behaviors in adolescents? 
Public health nutrition  2009;13(5):673-681.
Associations between diet and physical activity may identify behaviors that could be changed together to prevent childhood obesity. This study examines associations between physical activity and obesogenic dietary behaviors in a large UK youth cohort.
Cross-sectional analysis of UK cohort.
Subjects and methods
10–11 year old UK youths completed 3, one-day diet diaries. Average daily energy consumption, percent energy from fat, carbohydrate, energy density and grams of fruit and vegetables were estimated. To assess physical activity participants wore an accelerometer for 3 or more days. Regression models were run by sex to examine the extent to which dietary variables predicted physical activity before and after controlling for pubertal status, maternal education and adiposity.
Among boys percent energy from fat was consistently negatively associated with accelerometer determined indicators of physical activity (Std. Beta −.055 to −.101, p<.05) while total energy (Std. Beta = .066 to .091. p<.05) and percent energy from carbohydrate (.054 to .106, p<.05) were positively associated before and after adjustment for confounders. For girls fruit and vegetable intake was consistently positively associated with physical activity (Std. Beta = .056 to .074, p<.005). However all associations were weak. Associations were broadly comparable when participants with non-plausible dietary reports were included or excluded from the analyses.
Obesogenic diet and physical activity behaviors were weakly associated, suggesting that interventions should focus on implementing strategies that are independently successful at changing diet or physical activity behaviors either separately or in combination.
PMCID: PMC2913226  PMID: 19954571
ALSPAC; under-report; physical activity; childhood obesity
17.  Polygenic dissection of the bipolar phenotype 
The British Journal of Psychiatry  2011;198(4):284-288.
Recent data provide strong support for a substantial common polygenic contribution (i.e. many alleles each of small effect) to genetic susceptibility for schizophrenia and overlapping susceptibility for bipolar disorder.
To test hypotheses about the relationship between schizophrenia and psychotic types of bipolar disorder.
Using a polygenic score analysis to test whether schizophrenia polygenic risk alleles, en masse, significantly discriminate between individuals with bipolar disorder with and without psychotic features. The primary sample included 1829 participants with bipolar disorder and the replication sample comprised 506 people with bipolar disorder.
The subset of participants with Research Diagnostic Criteria schizoaffective bipolar disorder (n = 277) were significantly discriminated from the remaining participants with bipolar disorder (n = 1552) in both the primary (P = 0.00059) and the replication data-sets (P = 0.0070). In contrast, those with psychotic bipolar disorder as a whole were not significantly different from those with non-psychotic bipolar disorder in either data-set.
Genetic susceptibility influences at least two major domains of psychopathological variation in the schizophrenia–bipolar disorder clinical spectrum: one that relates to expression of a ‘bipolar disorder-like’ phenotype and one that is associated with expression of ‘schizophrenia-like’ psychotic symptoms.
PMCID: PMC3065773  PMID: 21972277
18.  Anatomical failure following laparoscopic antireflux surgery (LARS); does it really matter? 
Failure rates of laparoscopic antireflux surgery (LARS) vary from 2–30%. A degree of anatomical failure is common, and the most common failure is intrathoracic wrap herniation. We have assessed anatomical integrity of the crural repair and wrap using marking Liga clips placed at the time of surgery and compared this with symptomatic outcome.
A prospective study was undertaken on 50 patients who underwent LARS in a single centre over a 3-year period. Each had an X-ray on the first postoperative day and a barium swallow at 6 months at which the distance was measured between the marking Liga clips. An increase in interclip distance of > 25–49% was deemed ‘mild separation’, and an increase of > 50% ‘moderate separation’. Patients completed a standardised symptom questionnaire at 6 months.
At 6 months' postoperatively, 22% had mild separation of the crural repair with a mean Visick score of 1.18, and 54% had moderate separation with a mean Visick score of 1.26. Mild separation of the wrap occurred in 28% with a mean Visick score of 1.21 and 22% moderate separation with a mean Visick score of 1.18. Three percent had mild separation of both the crural repair and wrap with a mean Visick score of 1.0, and 16% moderate separation with a mean Visick score of 1.13. Of patients, 14% had evidence of some degree of failure on barium swallow but only one of these was significant intrathoracic migration of the wrap which was symptomatic and required re-do surgery.
The prevalence of some form of anatomical failure, as determined by an increase in the interclip distance, is high at 6 months' postoperatively following LARS. However, this does not seem to correlate with a subjective recurrence of symptoms.
PMCID: PMC3025249  PMID: 19995487
Laparoscopic antireflux surgery; LARS; Intrathoracic wrap herniation; Anatomical failure
19.  IE63-specific T-cell responses associate with control of subclinical varicella zoster virus reactivation in individuals with malignancies 
British Journal of Cancer  2010;102(4):727-730.
Reactivation of the varicella zoster virus (VZV) is more common in patients with malignancies; however, the molecular and cellular mechanisms underlying this susceptibility are unclear.
Using ex vivo interferon-γ ELISpot assays, we set out to analyse VZV-specific immune responses in a large cohort of patients with malignancies.
We observed that patients with malignancies had impaired VZV-specific T-cell responses, particularly in those with haematological malignancies and breast carcinoma. Immediate-early protein 63 (IE63)-specific T-cell responses were significantly impaired in those with subclinical VZV re-activation.
Our results suggest that T-cell responses to IE63 are important in controlling VZV replication.
PMCID: PMC2837573  PMID: 20087346
varicella zoster virus; malignancy; T-cell responses; IE63
20.  Effect of contact lens material on cytotoxicity potential of multipurpose solutions using human corneal epithelial cells 
Molecular Vision  2011;17:3458-3467.
Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses.
The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance.
A significant reduction in viability (between 30 to 50%) was observed with cells exposed to lenses soaked in ReNu and OFX. A significant downregulation of α3 and β1 integrins, with integrin expression ranging from 60% to 75% of control (cells with no lens), was also observed with OFX and ReNu-soaked lenses. With the exception of BA, all other lenses soaked in OFX resulted in significant caspase activation, whereby over 18% of cells stained positive for caspases. Minimal caspase activation was observed in cells exposed to ReNu and Solo soaked lenses. For both OFX and ReNu, exposing cells to at least a 5% dilution had a significant effect on viability and integrin expression. While Complete and Solo did not lead to reduction in viability, cells exposed to a 10% dilution showed reduced integrin expression down to less than 70% of control value. Comparing cell response to diluted MPS solutions and various MPS-soaked lenses showed that it is not possible to reliably use cell response to MPS dilution alone to assess MPS biocompatibility.
Our results demonstrate that the reaction of HCEC to MPS are affected by the type of lenses the MPS is released from and may potentially be influenced by the surface treatment (or lack of it) of SiHy materials.
PMCID: PMC3249433  PMID: 22219641
22.  Development and pilot evaluation of a complex intervention to improve experienced continuity of care in patients with cancer 
British Journal of Cancer  2008;100(2):274-280.
High experienced continuity of care in patients with cancer is associated with lower needs for care, better quality of life and better psychological outcomes. We developed and evaluated an intervention to improve experienced continuity. The intervention, consisted of (1) a 17-item patient-completed continuity assessment; (2) feedback to clinical nurse specialists and action to address the needs identified. Multidisciplinary team meetings and oncology outpatient clinics were observed, and patients and staff were interviewed. After qualitative work and reliability testing, the intervention was evaluated in a feasibility trial. Sixty-one patients provided data for analysis. No statistically significant differences were found in patients' experienced continuity between the trial arms, but important trends were seen in measures of needs for care in favour of those receiving the intervention. Feeding back findings from the continuity assessment to clinicians reduced patients' needs for care. Our results indicate that an intervention to target patients' experiences of continuity can reduce their subsequent needs for care. However, overcoming barriers to organisational change and addressing some patients' hesitation to report their continuity difficulties must be considered when implementing such an intervention. A phase III trial targeting patients with inadequate experienced continuity of care is recommended.
PMCID: PMC2634705  PMID: 19107130
continuity of care; complex intervention; feasibility trial
23.  Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions 
Molecular Vision  2010;16:272-282.
Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model.
An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate  (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis.
Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis.
Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells.
PMCID: PMC2823797  PMID: 20169012
24.  Single-Molecule Motions of Oligoarginine Transporter Conjugates on the Plasma Membrane of Chinese Hamster Ovary Cells 
To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the plasma membrane of Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by single-molecule diffusion coefficient and residence time (τR), defined as the time from the initial appearance of a single-molecule spot on the membrane (from the solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (amodel guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly heterogeneous and cannot be described as Brownian motion with a single diffusion coefficient. The observed behavior is also different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with cytochalasin D, a known actin filament disruptor, produces no significant effect, which further rules out unimodal endocytosis as the mechanism of uptake. Also, the involvement of membrane potential in octaarginine–membrane interaction is supported by significant changes in the motion with high [K+] treatment. In sum, this first study of single transporter motion on the membrane of a living cell indicates that the mode by which the octaarginine transporter penetrates the cell membrane appears to either be a multimechanism uptake process or a mechanism different from unimodal passive diffusion or endocytosis.
PMCID: PMC2725020  PMID: 18578528
25.  Paracetamol availability and recent changes in paracetamol poisoning: is the 1998 legislation limiting availability of paracetamol being followed? 
Postgraduate Medical Journal  2006;82(970):520-523.
To determine the degree of adherence to legislation introduced in 1998 restricting the availability of over the counter paracetamol.
A prospective observational study.
An emergency department in an inner city London teaching hospital. Pharmacy and non‐pharmacy outlets in south London.
Main outcome measures
(1) The source of paracetamol ingested by 107 patients presenting with an acute paracetamol overdose (2001–2003) and (2) the ability to purchase paracetamol from pharmacy and non‐pharmacy outlets in a manner contravening paracetamol pack size legislation (2004).
Potentially toxic amounts of paracetamol in excess of pack size restrictions were purchased in 70% (17 of 24) of outlets. Forty six per cent of patients who had ingested a potentially toxic dose of paracetamol obtained the tablets in a manner contravening the 1998 legislation.
Legislation limiting the availability of over the counter paracetamol is not being adhered to in south London. A significant number of patients ingesting a potentially toxic dose of paracetamol report purchasing the tablets in a manner contravening the legislation. Studies that attempt to assess the impact of the legislation need to be interpreted in the context of these results. Measures to enforce current legislation may help to reduce the severity of paracetamol poisoning in the UK.
PMCID: PMC2585716  PMID: 16891443
legislation; paracetamol; pack size; overdose

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