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1.  High levels of effective long-distance dispersal may blur ecotypic divergence in a rare terrestrial orchid 
BMC Ecology  2014;14:20.
Gene flow and adaptive divergence are key aspects of metapopulation dynamics and ecological speciation. Long-distance dispersal is hard to detect and few studies estimate dispersal in combination with adaptive divergence. The aim of this study was to investigate effective long-distance dispersal and adaptive divergence in the fen orchid (Liparis loeselii (L.) Rich.). We used amplified fragment length polymorphism (AFLP)-based assignment tests to quantify effective long-distance dispersal at two different regions in Northwest Europe. In addition, genomic divergence between fen orchid populations occupying two distinguishable habitats, wet dune slacks and alkaline fens, was investigated by a genome scan approach at different spatial scales (continental, landscape and regional) and based on 451 AFLP loci.
We expected that different habitats would contribute to strong divergence and restricted gene flow resulting in isolation-by-adaptation. Instead, we found remarkably high levels of effective long-distance seed dispersal and low levels of adaptive divergence. At least 15% of the assigned individuals likely originated from among-population dispersal events with dispersal distances up to 220 km. Six (1.3%) ‘outlier’ loci, potentially reflecting local adaptation to habitat-type, were identified with high statistical support. Of these, only one (0.22%) was a replicated outlier in multiple independent dune-fen population comparisons and thus possibly reflecting truly parallel divergence. Signals of adaptation in response to habitat type were most evident at the scale of individual populations.
The findings of this study suggest that the homogenizing effect of effective long-distance seed dispersal may overwhelm divergent selection associated to habitat type in fen orchids in Northwest Europe.
PMCID: PMC4099500  PMID: 24998243
2.  Testing the performance of a fragment of the COI gene to identify western Palaearctic stag beetle species (Coleoptera, Lucanidae) 
ZooKeys  2013;105-126.
The taxonomy of stag beetles (Coleoptera: Lucanidae) remains challenging, mainly due to the sexual dimorphism and the strong allometry in males. Such conjecture confounds taxonomic based conservation efforts that are urgently needed due to numerous threats to stag beetle biodiversity. Molecular tools could help solve the problem of identification of the different recognized taxa in the “Lucanus cervus complex” and in some related Palaearctic species. We investigated the potential use of a 670 bp region at the 3’ end of the mitochondrial cytochrome c oxidase subunit I gene (COI) for barcoding purposes (different from the standard COI barcoding region). Well resolved species and subspecies were L. tetraodon, L. cervusakbesianus, L. c. laticornis, as well as the two eastern Asian outgroup taxa L. formosanus and L. hermani. Conversely, certain taxa could not be distinguished from each other based on K2P-distances and tree topologies: L. c. fabiani / L. (P.) barbarossa, L. c. judaicus / an unknown Lucanus species, L. c. cervus / L. c. turcicus / L. c. pentaphyllus / L. (P.) macrophyllus / L. ibericus. The relative roles of phenotypic plasticity, recurrent hybridisation and incomplete lineage sorting underlying taxonomic and phylogenetic discordances are discussed.
PMCID: PMC3890674  PMID: 24453554
Lucanus spp.; Stag beetle; Western Palaearctic; DNA barcoding; COI
3.  Longitudinal, mixed method study to look at the experiences and knowledge of non melanoma skin cancer from diagnosis to one year 
BMC Dermatology  2013;13:13.
Skin cancer is the most common type of cancer in humans and the incidence is increasing worldwide. Our objective was to understanding the needs, experiences and knowledge of individuals with Non Melanoma Skin Cancer (NMSC) from diagnosis up until one year.
Patients with NMSC completed questionnaires at diagnosis, treatment, 8 weeks post treatment and 12 months post diagnosis. Body image, psychological morbidity and Quality of Life (QOL) were assessed at each time point, with the exception of QOL that was not assessed at diagnosis. Knowledge of NMSC was assessed at baseline and 8 weeks. A sub-sample of participants was also interviewed to allow a more in-depth exploration of patients’ experiences.
76 participants completed the initial questionnaire, of which 15 were interviewed. Patients were anxious about a diagnosis of skin cancer, however they were no more depressed or anxious than the general population. QOL significantly improved from diagnosis to 8 weeks and from diagnosis to one year. Knowledge of NMSC was poor and did not improve after treatment. Hairdressers were highlighted as playing an important role in raising awareness and encouraging individuals to seek medical help. Most participants were aware of the need to check their skin for suspicious lesions but were not sure what to look for. At one year participants had forgotten their experience and were not overly concerned about skin cancer.
There is a need to raise awareness of the signs and symptoms of NMSC. Information on skin cancer needs to be tailored to the individual both at the start of treatment and during the follow up months, ensuring that participants’ needs and expectations are met. Targeting education at individuals in the community who regularly come into contact with skin should help in early identification of NMSC. This is important since skin cancer caught early is easily treatable and delay in presentation leads to larger and more complex lesions which impacts in terms of increased morbidity and increased health care costs.
PMCID: PMC3819707  PMID: 24164857
Skin cancer; Non melanoma skin cancer; NMSC; Needs; Experiences; Knowledge
4.  Overexpression of BMP3 in the Developing Skeleton Alters Endochondral Bone Formation Resulting in Spontaneous Rib Fractures 
Bone morphogenetic protein-3 (BMP) has been identified as a negative regulator in the skeleton as mice lacking BMP3 have increased bone mass. To further understand how BMP3 mediates bone formation, we created transgenic mice overexpressing BMP3 using the type I collagen promoter. BMP3 transgenic mice displayed spontaneous rib fractures that were first detected at E17.0. The fractures were due to defects in differentiation of the periosteum and late hypertrophic chondrocytes resulting in thinner cortical bone with decreased mineralization. As BMP3 modulates BMP and activin signaling through ActRIIB, we examined the ribs of ActRIIB receptor knockout mice and found they had defects in late chondrogenesis and mineralization similar to BMP3 transgenic mice. These data suggest that BMP3 exerts its effects in the skeleton by altering signaling through ActRIIB in chondrocytes and the periosteum, and this results in defects in bone collar formation and late hypertrophic chondrocyte maturation leading to decreased mineralization and less bone.
PMCID: PMC3523674  PMID: 19653325
BMP3; ActRIIB; periosteum; ribs; chondrocytes
5.  After you: conversations between patients and healthcare professionals in planning for end of life care 
BMC Palliative Care  2012;11:15.
This study explores with patients, carers and health care professionals if, when and how Advance Care Planning conversations about patients’ preferences for place of care (and death) were facilitated and documented.
The study adopted an exploratory case study design using qualitative interviews, across five services delivering palliative care to cancer and non-cancer patients within an urban and rural English region. The study recruited 18 cases made up of patients (N = 18; 10 men; 8 women; median age 75); nominated relatives (N = 11; 7 women; 4 men; median age 65) and healthcare professionals (N = 15) caring for the patient. Data collection included: 18 initial interviews (nine separate interviews with patients and 9 joint interviews with patients and relatives) and follow up interviews in 6 cases (involving a total of 5 patients and 5 relatives) within one year of the first interview. Five group interviews were conducted with 15 healthcare professionals; 8 of whom also participated in follow up interviews to review their involvement with patients in our study.
Patients demonstrated varying degrees of reticence, evasion or reluctance to initiate any conversations about end of life care preferences. Most assumed that staff would initiate such conversations, while staff were often hesitant to do so. Staff-identified barriers included the perceived risks of taking away hope and issues of timing. Staff were often guided by cues from the patient or by intuition about when to initiate these discussions.
This study provides insights into the complexities surrounding the initiation of Advance Care Planning involving conversations about end of life care preferences with patients who are identified as having palliative care needs, in particular in relation to the risks inherent in the process of having conversations where mortality must be acknowledged. Future research is needed to examine how to develop interventions to help initiate conversations to develop person centred plans to manage the end of life.
PMCID: PMC3517317  PMID: 22985010
Advance care planning; Palliative care services; Preferred place of care; Qualitative research
6.  Comprehensive geriatric assessment for older women with early breast cancer – a systematic review of literature 
The Comprehensive Geriatric Assessment (CGA) is an analytical tool increasingly implemented in clinical practice. Breast cancer is primarily a disease of older people; however, most evidence-based research is aimed at younger patients.
A systematic review of literature was carried out to assess the use of CGA in older breast cancer patients for clinical decision making. The PubMed, Embase and Cochrane databases were searched.
A total of nine useful full text article results were found. Only five of these were exclusively concerned with early breast cancer; thus, studies involving a variety of cancer types, stages and treatments were accepted, as long as they included early breast cancer.The results comprised a series of low sources of evidence. However, all results shared a common theme: the CGA has a use in determining patient suitability for different types of cancer treatment and subsequently maximizing the patient’s quality of life.
There is not yet sufficient high level evidence to instate CGA guidelines as a mandatory practice in the management of breast cancer, due to the heterogeneity of available studies. More studies need to be conducted to cement current work on the benefits of the CGA. An area of particular interest is with regard to treatment options, especially surgery and chemotherapy, and identifying patients who may be suitable for these treatments.
PMCID: PMC3476996  PMID: 22594557
Comprehensive geriatric assessment; Primary; Operable; Breast cancer; Early
7.  External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds 
The Oncologist  2011;16(4):467-478.
The initial development of a regional KRAS external quality assessment scheme in 2009 in different European countries is described, providing a baseline picture of the accuracy and reliability of the analysis of the KRAS test to identify areas of particular difficulty in testing procedures and to assess the feasibility of a European external quality assessment scheme.
Learning Objectives
After completing this course, the reader will be able to: Identify the most frequent errors made in KRAS testing in this study and the possible consequences for a patient.Describe factors that could increase the chance of an error during KRAS testing.
This article is available for continuing medical education credit at
The use of epidermal growth factor receptor–targeting antibodies in metastatic colorectal cancer has been restricted to patients with wild-type KRAS tumors by the European Medicines Agency since 2008, based on data showing a lack of efficacy and potential harm in patients with mutant KRAS tumors. In an effort to ensure optimal, uniform, and reliable community-based KRAS testing throughout Europe, a KRAS external quality assessment (EQA) scheme was set up. The first large assessment round included 59 laboratories from eight different European countries. For each country, one regional scheme organizer prepared and distributed the samples for the participants of their own country. The samples included unstained sections of 10 invasive colorectal carcinomas with known KRAS mutation status. The samples were centrally validated by one of two reference laboratories. The laboratories were allowed to use their own preferred method for histological evaluation, DNA isolation, and mutation analysis. In this study, we analyze the setup of the KRAS scheme. We analyzed the advantages and disadvantages of the regional scheme organization by analyzing the outcome of genotyping results, analysis of tumor percentage, and written reports. We conclude that only 70% of laboratories correctly identified the KRAS mutational status in all samples. Both the false-positive and false-negative results observed negatively affect patient care. Reports of the KRAS test results often lacked essential information. We aim to further expand this program to more laboratories to provide a robust estimate of the quality of KRAS testing in Europe, and provide the basis for remedial measures and harmonization.
PMCID: PMC3228116  PMID: 21441573
Tumor markers; Genetic testing; Colorectal cancer; Quality assurance; KRAS
8.  FIRE (facilitating implementation of research evidence): a study protocol 
Research evidence underpins best practice, but is not always used in healthcare. The Promoting Action on Research Implementation in Health Services (PARIHS) framework suggests that the nature of evidence, the context in which it is used, and whether those trying to use evidence are helped (or facilitated) affect the use of evidence. Urinary incontinence has a major effect on quality of life of older people, has a high prevalence, and is a key priority within European health and social care policy. Improving continence care has the potential to improve the quality of life for older people and reduce the costs associated with providing incontinence aids.
This study aims to advance understanding about the contribution facilitation can make to implementing research findings into practice via: extending current knowledge of facilitation as a process for translating research evidence into practice; evaluating the feasibility, effectiveness, and cost-effectiveness of two different models of facilitation in promoting the uptake of research evidence on continence management; assessing the impact of contextual factors on the processes and outcomes of implementation; and implementing a pro-active knowledge transfer and dissemination strategy to diffuse study findings to a wide policy and practice community.
Setting and sample
Four European countries, each with six long-term nursing care sites (total 24 sites) for people aged 60 years and over with documented urinary incontinence
Methods and design
Pragmatic randomised controlled trial with three arms (standard dissemination and two different programmes of facilitation), with embedded process and economic evaluation. The primary outcome is compliance with the continence recommendations. Secondary outcomes include proportion of residents with incontinence, incidence of incontinence-related dermatitis, urinary tract infections, and quality of life. Outcomes are assessed at baseline, then at 6, 12, 18, and 24 months after the start of the facilitation interventions. Detailed contextual and process data are collected throughout, using interviews with staff, residents and next of kin, observations, assessment of context using the Alberta Context Tool, and documentary evidence. A realistic evaluation framework is used to develop explanatory theory about what works for whom in what circumstances.
Trial registration
Current Controlled Trials ISRCTN11598502.
PMCID: PMC3356232  PMID: 22453077
9.  Enhancing communication in oncology outpatient consultations: critical reflections from doctors 
The experiences of patients diagnosed with advanced incurable cancer and the doctors who conducted their medical consultations were studied in order to improve the understanding of what happens in consultations, when bad news is disclosed. The major objective of the study was to critically reflect upon doctor-patient communication, in such situations, with a view to considering future strategies for doctors’ continuing professional development.
Sixteen patients and sixteen Oncologists, from a cancer centre in the UK were recruited into this ethnographic study. One hundred and fifteen episodes of data were collected from audio recorded consultations; interviews with doctors and patients and their relatives and observations of consultations. These data were analysed using a constant comparison method.
Interactions between doctors and patients are complex and consultations can be challenging for both of them. Some doctors spoke openly about their need for additional support to enhance their communication related competencies within Oncology consultations. These doctors wanted to observe their peers conducting consultations. They also wanted to receive feedback about their own clinical practices. These doctors stated that they wanted an open culture whereby they could talk freely about difficult and emotionally challenging consultations without fear of being considered incompetent by their Consultants, who act in a clinical supervisory role.
To help practitioners consolidate their practice in such settings it is necessary to develop better collaborations among practitioners within clinical practice. Providing individual supervisory sessions or group workshops can facilitate reflective learning and provide an open and supportive learning culture.
PMCID: PMC4205512
Communication; reflection; continued professional development
10.  Is it recorded in the notes? Documentation of end-of-life care and preferred place to die discussions in the final weeks of life 
BMC Palliative Care  2011;10:18.
Over the past ten years there has been an increasing focus on the need for improving the experience of end of life care. A number of policy initiatives have been introduced to develop approaches to discussing and documenting individual preferences for end of life care, in particular preferred place to die.
The aim was to investigate practice in relation to discussing and documenting end of life care and preferred place to die in the last 4 weeks of life with patients and their families. The study utilised an audit of 65 case notes, alongside four group interviews with a mix of health care professionals involved in palliative care provision.
While there was evidence that discussions relating to end of life care and preferred place to die had taken place in around half of the audited case notes, there appeared to be a lack of a systematic approach to the recording of discussions with patients or carers about these kind of issues. Health care staff subsequently highlighted that initiating discussions about end of life care and preferences in relation to place of death was challenging and that the recording and tracking of such preferences was problematic.
Further work is required to establish how information may be adequately recorded, revised and transferred across services to ensure that patients' preferences in relation to end of life care and place of death are, as far as possible, achieved.
PMCID: PMC3227605  PMID: 22053810
11.  Genetic structure and seed-mediated dispersal rates of an endangered shrub in a fragmented landscape: a case study for Juniperus communis in northwestern Europe 
BMC Genetics  2011;12:73.
Population extinction risk in a fragmented landscape is related to the differential ability of the species to spread its genes across the landscape. The impact of landscape fragmentation on plant population dynamics will therefore vary across different spatial scales. We quantified successful seed-mediated dispersal of the dioecious shrub Juniperus communis in a fragmented landscape across northwestern Europe by using amplified fragment length polymorphism (AFLP) markers. Furthermore we investigated the genetic diversity and structure on two spatial scales: across northwestern Europe and across Flanders (northern Belgium). We also studied whether seed viability and populations size were correlated with genetic diversity.
Unexpectedly, estimated seed-mediated dispersal rates were quite high and ranged between 3% and 14%. No population differentiation and no spatial genetic structure were detected on the local, Flemish scale. A significant low to moderate genetic differentiation between populations was detected at the regional, northwest European scale (PhiPT = 0.10). In general, geographically nearby populations were also genetically related. High levels of within-population genetic diversity were detected but no correlation was found between any genetic diversity parameter and population size or seed viability.
In northwestern Europe, landscape fragmentation has lead to a weak isolation-by-distance pattern but not to genetic impoverishment of common juniper. Substantial rates of successful migration by seed-mediated gene flow indicate a high dispersal ability which could enable Juniperus communis to naturally colonize suitable habitats. However, it is not clear whether the observed levels of migration will suffice to counterbalance the effects of genetic drift in small populations on the long run.
PMCID: PMC3176195  PMID: 21859457
12.  Feedback of trial results to participants: A survey of clinicians’ and patients’ attitudes and experiences☆ 
European Journal of Oncology Nursing  2011;15(2-4):124-129.
There is growing interest in the provision of trial results to trial participants. However, there are a number of gaps in the research base relating to the closure of clinical trials and feedback of results to participants.
The aim of this research was to explore the practice of feeding back trial results to trial participants and to identify best practice in this area. Postal questionnaires were sent to members of the UK National Cancer Research Institute Clinical Studies Groups (NCRI CSG) and to patients over the age of 18 years who completed trial treatment (located in one Cancer Network) during a 16-month period (April 07–July 08).
145 NCRI CSG member surveys and 81 patient questionnaires were returned. The vast majority of all respondents supported the idea of offering results to trial participants. However, NCRI members and trial participants differed in their opinions about the timing and method for the provision of results.
The results provide an insight into the views of these groups in relation to desire for results and practical aspects of results feedback which should inform further investigations into trial management and the practice of feedback of trial results.
PMCID: PMC3078333  PMID: 20674498
Clinical Trials; Cancer; Results; Feedback
13.  "Did the trial kill the intervention?" experiences from the development, implementation and evaluation of a complex intervention 
The development, implementation and evaluation of any new health intervention is complex. This paper uses experiences from the design, implementation and evaluation of a rehabilitation programme to shed light on, and prompt discussion around, some of the complexities involved in such an undertaking.
Semi-structured interviews were conducted with 15 trial participants and five members of staff at the conclusion of a trial evaluating a rehabilitation programme aimed at promoting recovery after stem cell transplantation.
This study identified a number of challenges relating to the development and evaluation of complex interventions. The difficulty of providing a standardised intervention that was acceptable to patients was highlighted in the participant interviews. Trial participants and some members of staff found the concept of equipoise and randomisation challenging and there was discord between the psychosocial nature of the intervention and the predominant bio-medical culture in which the research took place.
A lack of scientific evidence as to the efficacy of an intervention does not preclude staff and patients holding strong views about the benefits of an intervention. The evaluation of complex interventions should, where possible, facilitate not restrict that complexity. Within the local environment where the trial is conducted, acquiescence from those in positions of authority is insufficient; commitment to the trial is required.
PMCID: PMC3056847  PMID: 21362159
14.  Expression and Function of BMP3 During Chick Limb Development 
Bone morphogenetic proteins (BMPs) play diverse roles in many aspects of skeletal development and bone homeostasis. During endochondral ossification, tight regulation of BMP activity is required to assure proper survival, proliferation and differentiation of skeletal progenitor cells into chondrocytes and osteoblasts. BMP3, a structurally divergent member of the BMP family, acts as a negative regulator of bone formation by limiting BMP signal transduction. In this study, we focus on the chick limb where we find BMP3 has a unique localization pattern with strong expression in the developing perichondrium. Overexpression of BMP3 in chick wing bud at the onset of chondrogenesis, using replication competent retrovirus, reduces BMP signaling leading to increased cell proliferation and delayed cell differentiation, resulting in expanded skeletal elements and joint fusions. Our results suggest that BMP3 expression in the perichondrium may serve to regulate cartilage cell proliferation by modulating the levels of BMP signaling, thus ensuring proper endochondral ossification.
PMCID: PMC2862365  PMID: 18489005
BMP; BMP3; ActRIIB; perichondrium; chick limb
15.  Conditional Deletion of BMP7 from the Limb Skeleton Does Not Affect Bone Formation or Fracture Repair 
While the osteoinductive activity of recombinant bone morphogenetic protein 7 (BMP7) is well established, evaluation of the role of endogenous BMP7 in bone formation and fracture healing has been hampered by perinatal lethality in BMP7 knockout mice. Here we employ conditional deletion of BMP7 from the embryonic limb prior to the onset of skeletogenesis to create limb bones lacking BMP7. We find that the absence of locally produced BMP7 has no effect on postnatal limb growth, articular cartilage formation, maintenance of bone mass, or fracture healing. Our data suggest that other BMPs present in adult bone are sufficient to compensate for the absence of BMP7.
PMCID: PMC2862367  PMID: 19780203
BMP7; conditional deletion; fracture repair
16.  Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth 
The Journal of biological chemistry  2006;281(37):27481-27491.
Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophy. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of cardiac working load. However, the functional significance of this biological event have not been determined, mainly due to the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (αMHC-BMP10) in which the cardiac specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These αMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. αMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in αMHC-BMP10 mice was solely due to defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the response of these hearts to both physiological (e.g., exercise-induced hypertrophy) and pathological hypertrophic stimuli remained normal. In addition, the αMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by 4 months of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophy.
PMCID: PMC2628764  PMID: 16798733
17.  Circulating Markers Reflect Both Anti- and Pro-Atherogenic Drug Effects in ApoE-Deficient Mice 
Biomarker Insights  2008;3:147-157.
Current drug therapy of atherosclerosis is focused on treatment of major risk factors, e.g. hypercholesterolemia while in the future direct disease modification might provide additional benefits. However, development of medicines targeting vascular wall disease is complicated by the lack of reliable biomarkers. In this study, we took a novel approach to identify circulating biomarkers indicative of drug efficacy by reducing the complexity of the in vivo system to the level where neither disease progression nor drug treatment was associated with the changes in plasma cholesterol.
ApoE−/− mice were treated with an ACE inhibitor ramipril and HMG-CoA reductase inhibitor simvastatin. Ramipril significantly reduced the size of atherosclerotic plaques in brachiocephalic arteries, however simvastatin paradoxically stimulated atherogenesis. Both effects occurred without changes in plasma cholesterol. Blood and vascular samples were obtained from the same animals. In the whole blood RNA samples, expression of MMP9, CD14 and IL-1RN reflected pro-and anti-atherogenic drug effects. In the plasma, several proteins, e.g. IL-1β, IL-18 and MMP9 followed similar trends while protein readout was less sensitive than RNA analysis.
In this study, we have identified inflammation-related whole blood RNA and plasma protein markers reflecting anti-atherogenic effects of ramipril and pro-atherogenic effects of simwastatin in a mouse model of atherosclerosis. This opens an opportunity for early, non-invasive detection of direct drug effects on atherosclerotic plaques in complex in vivo systems.
PMCID: PMC2688353  PMID: 19578502
18.  Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity 
The Journal of Clinical Investigation  2007;117(9):2638-2648.
Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.
PMCID: PMC1957541  PMID: 17786246
19.  The Way that PEGyl-DSPC Liposomal Doxorubicin Particles Penetrate into Solid Tumor Tissue 
Drug Target Insights  2006;1:29-34.
For enhancement of drug effectiveness and reduction of drug toxicity, liposomal drugs have been studied in laboratories and clinics for decades. Although the results obtained from in vitro are encouraging, but the results from in vivo tests were not satisfactory. The main reasons for this situation were that we do not have enough information about the way how liposomal particles penetrating into solid tumor tissue, and what happening to the liposome particles after they got into the tumor tissue. In this paper, we are going to report the results from our observations on the way folic acid targeted and non-targeted PEGyl-DSPC liposomal doxorubicin particles penetrate into solid tumor tissue.
Subcutaneous transplanted murine L1210JF solid tumors in mice were used as a model. PEGyl liposomal doxorubicins were injected through tail venue, and tumor tissue samples were collected at special time points. Cryosections were cut and dried by a flowing of air after mounted on the slides right away. Then the dried cryosections were stained in water systems; the blood vessel cells were stained with green fluorescent FITC labeled antibody against CD31 antigen; the nuclei of the living cells were stained with a blue fluorescent dye DAPI. Since the whole procedure was carried out in aquatic system, the red color fluorescent liposomal doxorubicin particles remain visible under fluorescence microscope.
Both folate conjugated and non-conjugated PEGyl-DSPC liposomal doxorubicin particles were only leaking out from the broken holes of blood vessels with a special direction and spread out for a limited distance, which was similar to the results showed before, in that observation a latex microsphere sample was used as a model.
PMCID: PMC3155213  PMID: 21901056
Solid tumor; liposome particles; blood vessel penetration
20.  Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis 
PLoS Genetics  2006;2(12):e216.
Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.
A group of related signaling molecules called bone morphogenetic proteins (BMPs) are known to play important roles in the formation of the structures such as the limbs. However, because different members of this group often have similar effects on target cells and are produced in overlapping regions of the embryo and hence can be redundant with one another, removal of any single member of the BMP family may not reveal the full extent of the roles they play during development. We have therefore improved on this type of analysis by removing pairs of these factors (BMP2 and BMP4 or BMP2 and BMP7) specifically from the developing limb. Although some have speculated that these signals play an early role in organizing or “patterning” the different tissues of the limb, we find no evidence for such a role. We do find, however, that a minimal amount of BMP signal is required to form cartilage, and hence some cartilaginous elements fail to form in limbs deficient in both BMP2 and BMP4. Moreover, in the absence of these two BMP family members, there is a severe impairment in the development of bone tissue, resulting in severely deformed limbs. This study gives important new insight into the roles of these BMP signals in making skeletal tissues in the embryo.
PMCID: PMC1713256  PMID: 17194222
21.  The heat-stable antigen determines pathogenicity of self-reactive T cells in experimental autoimmune encephalomyelitis 
Journal of Clinical Investigation  2000;105(9):1227-1232.
Induction of myelin-specific CD4 T cells is a pivotal event in the development of experimental autoimmune encephalomyelitis (EAE). Other checkpoints in EAE pathogenesis have not been clearly defined, although multiple genetic loci are known to influence EAE development. We report here that targeted mutation of the heat-stable antigen (HSA) abrogates development of EAE despite a complete lack of effect on induction of autoimmune T cells. To test whether T-cell expression of HSA is sufficient, we created transgenic mice in which HSA is expressed exclusively in the T-cell lineage. We found that these mice remain resistant to EAE induction. Adoptive transfer studies demonstrate that both T cells and non–T cells must express HSA in order for the pathogenic T cells to execute their effector function. Moreover, HSAIg, a fusion protein consisting of the extracellular domain of the HSA and the Fc portion of immunoglobulin, drastically ameliorates the clinical sign of EAE even when administrated after self-reactive T cells had been expanded. Thus, identification of HSA as a novel checkpoint, even after activation and expansion of self-reactive T cells, provides a novel approach for immunotherapy of autoimmune neurologic diseases, such as multiple sclerosis.
PMCID: PMC315444  PMID: 10791997

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