Neoplasia in Three Aye-Ayes (Daubentonia madagascariensis).

Tumours diagnosed in three aged captive aye-ayes (Daubentonia madagascariensis), held in two different institutions, are described. A cerebral glioblastoma was diagnosed based on histological and immunohistochemical findings in one of the animals following initial presentation with bilateral mydriasis, absent pupillary reflex, head tilt and ataxia. A second animal was humanely destroyed due to impaired locomotion associated with spondylosis and a post-mortem diagnosis of cholangiocarcinoma was made based on histology with further confirmation with immunohistochemical labelling for cytokeratin 7. A third aye-aye suffering from dental disease was diagnosed with an oral squamous cell carcinoma following an excisional biopsy from a non-healing wound in the lip. Due to progression of the neoplasia the animal was humanely destroyed and post-mortem examination revealed the presence on an additional unilateral phaeochromocytoma.

Assessing pain objectively: the use of physiological markers.

Pain diagnosis and management would benefit from the development of objective markers of nociception and pain. Current research addressing this issue has focused on five main strategies, each with its own advantages and disadvantages. These encompass: (i) monitoring changes in the autonomic nervous system; (ii) biopotentials; (iii) neuroimaging; (iv) biological (bio-) markers; and (v) composite algorithms. Although each strategy has shown areas of promise, there are currently no validated objective markers of nociception or pain that can be recommended for clinical use. This article introduces the most important developments in the field and highlights shortcomings, with the aim of allowing the reader to make informed decisions about what trends to watch in the future.

Expression of hypoxia-inducible factor 1 alpha in thyroid carcinomas.

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is upregulated by hypoxia and oncogenic signalling in many solid tumours. Its regulation and function in thyroid carcinomas are unknown. We evaluated the regulation of HIF-1 alpha and target gene expression in primary thyroid carcinomas and thyroid carcinoma cell lines (BcPAP, WRO, FTC-133 and 8505c). HIF-1 alpha was not detectable in normal tissue but was expressed in thyroid carcinomas. Dedifferentiated anaplastic tumours (ATCs) exhibited high levels of nuclear HIF-1 alpha staining. The HIF-1 target glucose transporter 1 was expressed to a similar level in all tumour types, whereas carbonic anhydrase-9 was significantly elevated in ATCs. In vitro studies revealed a functionally active HIF-1 alpha pathway in thyroid cells with transcriptional activation observed after graded hypoxia (1% O(2), anoxia) or treatment with a hypoxia mimetic cobalt chloride. High basal and hypoxia-induced expression of HIF-1 alpha in FTC-133 cells that harbour a phosphatase and tensin homologue (PTEN) mutation was reduced by introduction of wild-type PTEN. Similarly, pharmacological inhibition of the phosphoinositide 3-kinase (PI3K) pathway using LY294002 inhibited HIF-1 alpha and HIF-1 alpha targets in all cell lines, including those with B-RAF mutations (BcPAP and 8505c). In contrast, the effects of inhibition of the RAF/MEK/extracellular signal-regulated kinase pathway were restricted by environmental condition and B-RAF mutation status. HIF-1 is functionally expressed in thyroid carcinomas and is regulated not only by hypoxia but also via growth factor signalling pathways and, in particular, the PI3K pathway. Given the strong association of HIF-1 alpha with an aggressive disease phenotype and therapeutic resistance, this pathway may be an attractive target for improved therapy in thyroid carcinomas.

Contribution of HIF-1 and drug penetrance to oxaliplatin resistance in hypoxic colorectal cancer cells.

BACKGROUND: Hypoxia is as an indicator of poor treatment outcome. Consistently, hypoxic HCT116 colorectal cancer cells are resistant to oxaliplatin, although the mechanistic basis is unclear. This study sought to investigate the relative contribution of HIF-1 (hypoxia-inducible factor-1)-mediated gene expression and drug penetrance to oxaliplatin resistance using three-dimensional spheroids. METHODS: Hypoxia-inducible factor-1alpha function was suppressed by the stable expression of a dominant-negative form in HCT116 cells (DN). Cells were drug exposed as monolayer or multicellular spheroid cultures. Cells residing at differing oxygenation status were isolated from Hoechst 33342-treated spheroids using flow cytometry. Sub-populations were subjected to clonogenic survival assays and to Inductively-Coupled Plasma Mass Spectroscopy to determine oxaliplatin uptake. RESULTS: In spheroids, a sensitivity gradient (hypoxic

Population structure in a common Caribbean coral-reef fish: implications for larval dispersal and early life-history traits.

Genetic population structure throughout the Caribbean Basin for one of the most common and widespread reef fish species, the bicolour damselfish Stegastes partitus was examined using microsatellite DNA markers. Spatial autocorrelation analysis showed a significant positive correlation between genetic and geographic distance (isolation by distance) over distances <1000 km, suggesting that populations are connected genetically but probably not demographically, i.e. over shorter time scales. A difference in spatial patterns of populations in the eastern v. the western Caribbean also raises the probability of an important role for meso-scale oceanographic features and landscape complexity within the same species. A comparison of S. partitus population structure and life-history traits with those of two other species of Caribbean reef fish studied earlier showed the findings to be concordant with a common hypothesis that shorter pelagic larval dispersal periods are associated with smaller larval dispersal scales.

Small-scale demographic variation in the stoplight parrotfish Sparisoma viride.

Age-based analysis of the stoplight parrotfish Sparisoma viride was used to examine whether observed differences in their abundance and size structure among reefs in a cross-shelf portion of the upper Florida Keys could be explained by variation in demographic rates. Annual and daily sagittal otolith increments were enumerated for 176 individuals collected from replicates of reefs in two strata, inshore and offshore reefs (2-6 m depth). von Bertalanffy growth functions fitted to size-at-age plots for each site were similar between reefs within each stratum (inshore and offshore), but differed between strata. Sparisoma viride on offshore reefs attained greater average standard length (L(S)) at age, greater mean asymptotic size and were longer lived than fish from inshore reefs. Fish on inshore reefs attained only half the maximum age observed on offshore reefs (4 v. 8 years, respectively). No terminal phase fish >4 years of age were found on either reef type. Estimates of mortality rates from age-frequency data of collected fish revealed higher mortality on inshore reefs. Demographic variables obtained in this study were similar to published values for S. viride from Caribbean reefs but differed significantly from published values from reefs at a similar latitude (Bahamas), reflecting high demographic plasticity on both local and regional scales.

Gene therapy approaches to enhance bioreductive drug treatment.

Hypoxia, or a lack of oxygen, occurs in 50-60% of solid human tumours. Clinical studies have shown that the presence and extent of hypoxia in a tumour cannot be predicted by size or histopathological stage but it is predictive of a poor outcome following radiotherapy, chemotherapy and surgery. However, as a physiological feature of tumours, it can be exploited and researchers have developed many hypoxia-selective chemotherapies or bioreductive drugs that are in varying stages of clinical development. These agents are prodrugs that have two key requirements for their biological activation: they require the reductive environment of a hypoxic tumour cell and the appropriate complement of cellular reductase enzymes. To overcome tumour heterogeneity in reductase enzyme levels and enhance bioreductive drug metabolism a gene therapy strategy can be employed. We have reviewed this field and also present our own pre-clinical research using gene therapy to enhance bioreductive drug treatment for the treatment of cancer. We have specifically focused on studies enhancing lead clinical bioreductive drugs. We consider the metabolic requirements for their activation and we highlight the key in vivo studies supporting the future clinical development of hypoxia-targeted gene-directed enzyme prodrug therapy.

The acute blue finger: management and outcome.

INTRODUCTION: The objective was to assess the management, and short- and longer-term outcome of patients presenting with an acute blue finger. PATIENTS AND METHODS: This was a retrospective, case-note review and prospective follow-up by telephone and general practitioner enquiry. All patients who presented with sudden onset blue discolouration of a finger within the previous 72 h, with normal radial and ulnar pulses, were included. RESULTS: From 2000 to 2006, 22 patients, 15 female, 7 male, were reviewed. Median age was 56 years (range, 19-88 years). Median time from onset of blue finger was 6 days (range 1 day to 3 months). In most cases (17), no underlying cause was identified. Five patients had an underlying cause; two had symptoms compatible with Raynaud's phenomenon, one patient had signs (later confirmed on MRA) of arterial thoracic outlet syndrome and two had polycythaemia (haemoglobin > 17 g/dl). Otherwise, all laboratory investigations were normal. Upper limb duplex, echocardiogram and 24-h cardiac tapes were normal in all cases. Median follow-up was 19 months. Three patients had recurrent symptoms in the finger. No patient suffered tissue loss or loss of digit(s), and none had stroke or arterial embolisation. CONCLUSIONS: The acute blue finger is a benign condition not suggestive of arterial embolisation. Tissue or digit loss is not a threat and, in the longer term, there is no threat of embolisation to other vascular sites.

Bioreductive drugs: from concept to clinic.

One of the key issues for radiobiologists is the importance of hypoxia to the radiotherapy response. This review addresses the reasons for this and primarily focuses on one aspect, the development of bioreductive drugs that are specifically designed to target hypoxic tumour cells. Four classes of compound have been developed since this concept was first proposed: quinones, nitroaromatics, aliphatic and heteroaromatic N-oxides. All share two characteristics: (1) they require hypoxia for activation and (2) this activation is dependent on the presence of specific reductases. The most effective compounds have shown the ability to enhance the anti-tumour efficacy of agents that kill better-oxygenated cells, i.e. radiation and standard cytotoxic chemotherapy agents such as cisplatin and cyclophosphamide. Tirapazamine (TPZ) is the most widely studied of the lead compounds. After successful pre-clinical in vivo combination studies it entered clinical trial; over 20 trials have now been reported. Although TPZ has enhanced some standard regimens, the results are variable and in some combinations toxicity was enhanced. Banoxantrone (AQ4N) is another agent that is showing promise in early phase I/II clinical trials; the drug is well tolerated, is known to locate in the tumour and can be given in high doses without major toxicities. Mitomycin C (MMC), which shows some bioreductive activation in vitro, has been tested in combination trials. However, it is difficult to assign the enhancement of its effects to targeting of the hypoxic cells because of the significant level of its hypoxia-independent toxicity. More specific analogues of MMC, e.g. porfiromycin and apaziquone (EO9), have had variable success in the clinic. Other new drugs that have good pre-clinical profiles are PR 104 and NLCQ-1; data on their clinical safety/efficacy are not yet available. This paper reviews the pre-clinical data and discusses the clinical studies that have been reported.

Scaling of connectivity in marine populations.

Defining the scale of connectivity, or exchange, among marine populations and determining the factors driving this exchange are pivotal to our understanding of the population dynamics, genetic structure, and biogeography of many coastal species. Using a high-resolution biophysical model for the Caribbean region, we report that typical larval dispersal distances of ecologically relevant magnitudes are on the scale of only 10 to 100 kilometers for a variety of reef fish species. We also show the importance of the early onset of active larval movement mediating the dispersal potential. In addition to self-recruitment, larval import from outside the local area is required to sustain most populations, although these population subsidies are very limited in particular systems. The results reveal distinct regions of population isolation based on larval dispersal that also correspond to genetic and morphological clines observed across a range of marine organisms.

Current and future strategies for the treatment of malignant brain tumors.

Glioblastoma (GB) is the most common subtype of primary brain tumor in adults. These tumors are highly invasive, very aggressive, and often infiltrate critical neurological areas within the brain. The mean survival time after diagnosis of GB has remained unchanged during the last few decades, in spite of advances in surgical techniques, radiotherapy, and also chemotherapy; patients' survival ranges from 9 to 12 months after initial diagnosis. In the same time frame, with our increasing understanding and knowledge of the physiopathology of several cancers, meaningful advances have been made in the treatment and control of several cancers, such as breast, prostate, and hematopoietic malignancies. Although a number of the genetic lesions present in GB have been elucidated and our understanding of the progressions of this cancer has increased dramatically over the last few years, it has not yet been possible to harness this information towards developing effective cures. In this review, we will focus on the classical ways in which GB is currently being treated, and will introduce a novel therapeutic modality, i.e., gene therapy, which we believe will be used in combination with classical treatment strategies to prolong the life-span of patients and to ultimately be able to control and/or cure these brain tumors. We will discuss the use of several vector systems that are needed to introduce the therapeutic genes within either the tumor mass, if these are not resectable, or the tumor bed, after successful tumor resection. We also discuss different therapeutic modalities that could be exploited using gene therapy, i.e., conditional cytotoxic approach, direct cytotoxicity, immunotherapy, inhibition of angiogenesis, and the use of pro-apoptotic genes. The advantages and disadvantages of each of the current vector systems available to transfer genes into the CNS are also discussed. With the advances in molecular techniques, both towards the elucidation of the physiopathology of GB and the development of novel, more efficient and less toxic vectors to deliver putative therapeutic genes into the CNS, it should be possible to develop new rationale and effective therapeutic approaches to treat this devastating cancer.

Oxygen-sensitive enzyme-prodrug gene therapy for the eradication of radiation-resistant solid tumours.

Overwhelming clinical and experimental data demonstrate that tumour hypoxia is associated with aggressive disease and poor treatment outcome as hypoxic cells are refractive to radiotherapy and some forms of chemotherapy. However, hypoxia is rare in physiologically normal tissues representing a tumour-specific condition. To selectively target this therapeutically refractive cell population, we have combined bioreductive chemotherapy with hypoxia-directed gene therapy. We have transfected the human fibrosarcoma cell line, HT1080, with a hypoxia-regulated expression vector encoding the human flavoprotein cytochrome c P450 reductase (HRE-P450R). This conferred hypoxia-dependent sensitivity to the alkylating nitroimidazole prodrug RSU1069 in vitro, with a greater than 30-fold increase in oxic/hypoxic cytotoxicity ratio compared with controls. Xenografts of both the HRE-P450R and empty vector transfectants had comparable hypoxic fractions and were refractive to single dose radiotherapy of up to 15 Gy. However, combining a prodrug of RSU1069 with a reduced radiotherapy dose of 10 Gy represents a curative regimen (50% tumour-free survival; day 100) in the HRE-P450R xenografts. In complete contrast, 100% mortality was apparent by day 44 in the empty vector control xenografts treated in the same way. Thus, an oxygen-sensitive gene-directed enzyme prodrug therapy approach may have utility when incorporated into conventional radiotherapy and/or chemotherapy protocols for loco-regional disease in any tissue where hypoxia is a contra-indication to treatment success. doi:10.1038/sj.gt.3301702

Hypoxia and oxidative stress. Tumour hypoxia--therapeutic considerations.

Conclusive research has shown that regions of acute/chronic hypoxia, which exist within the majority of solid tumours, have a profound influence on the therapeutic outcome of cancer chemotherapy and radiotherapy and are a strong prognostic factor of disease progression and survival. A strong argument therefore exists for assessing the hypoxic fraction of tumours, prior to patient treatment, and to tailor this treatment accordingly. Tumour hypoxia also provides a powerful physiological stimulus that can be exploited as a tumour-specific condition, allowing for the rationale design of hypoxia-activated anticancer drugs or novel hypoxia-regulated gene therapy strategies.

Gene therapy strategies for intracranial tumours: glioma and pituitary adenomas.

Intracranial tumours such as brain gliomas and pituitary adenomas pose a challenging area of research for the development of gene therapy strategies, both from the point of view of the severity of the diseases, to the physiological implication of gene delivery into the central nervous system and pituitary gland. On the one hand, brain gliomas are very malignant tumours, with a life expectancy of six months to a year at the most after the time of diagnosis, in spite of advances in treatment modalities which involve chemotherapy, surgery and radiotherapy. Gene therapy for these tumours is therefore a very attractive therapeutic modality which due to the severity of the disease is already in clinical trials. On the other hand, pituitary tumours are usually benign, and in most cases, treatment is successful. Nevertheless, there are some instances, especially with the macroadenomas and some invasive tumours in which treatment fails. Gene therapy strategies for these adenomas therefore needs to progress substantially in terms of safety, adverse side effects and physiological impact on the normal pituitary gland before clinical implementation. In this paper, we will review gene delivery systems both viral and non-viral and several therapeutic strategies which could be implemented for the treatment of these diseases. These include cytotoxic approaches both conditional and direct, immune-stimulatory strategies, anti-angiogenic strategies and approaches which harness pro-apoptotic and tumour suppressor gene targets. We will also review the models which are currently available in which these gene therapy strategies can be tested experimentally. This new therapeutic modality holds enormous promise, but we still need substantial improvements both from the delivery, efficacy and safety stand points before it can become a clinical reality.

Connectivity of marine populations: open or closed?

Most marine populations are thought to be well connected via long-distance dispersal of larval stages. Eulerian and Lagrangian flow models, coupled with linear mortality estimates, were used to examine this assumption. The findings show that when simple advection models are used, larval exchange rates may be overestimated; such simplistic models fail to account for a decrease of up to nine orders of magnitude in larval concentrations resulting from diffusion and mortality. The alternative process of larval retention near local populations is shown to exist and may be of great importance in the maintenance of marine population structure and management of coastal marine resources.

Connectivity of marine populations: open or closed?

Most marine populations are thought to be well connected via long-distance dispersal of larval stages. Eulerian and Lagrangian flow models, coupled with linear mortality estimates, were used to examine this assumption. The findings show that when simple advection models are used, larval exchange rates may be overestimated; such simplistic models fail to account for a decrease of up to nine orders of magnitude in larval concentrations resulting from diffusion and mortality. The alternative process of larval retention near local populations is shown to exist and may be of great importance in the maintenance of marine population structure and management of coastal marine resources.(Au)

Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidylinositol (GPI) membrane anchor in neurons and polarised epithelial cells.

The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector-mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosyl-phosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell-cell interactions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing its cell killing effects.

Ichthyological enthusiasm.

Coral Reef Fishes: Caribbean, Indian Ocean, and Pacific Ocean including the Red Sea by E. Lieske and R. Myers Princeton University Press, 1996. $35.00 hbk (400 pages) ISBN 0 691 02659 9 Air-Breathing Fishes: Evolution, Diversity, and Adaptation by J.B. Graham Academic Press, 1997. $79.95 hbk (xi+299 pages) ISBN 0 12 294860 2 The Diversity of Fishes by G.S. Helfman, B.B. Collette and D.E. Facey Blackwell, 1997. £55.00 hbk (xii+528 pages) ISBN 0 86542 256 7.

Physical localisation of the breakpoints of a constitutional translocation t(5;6)(q21;q21) in a child with bilateral Wilms' tumour.

A 6 month old boy presented with bilateral Wilms' tumour. Cytogenetic analysis of the lymphocytes from the patient showed a de novo balanced translocation t(5;6)(q21;q21), which was also present in the tumour material as the sole cytogenetic abnormality. To facilitate the identification of the translocation breakpoints, we have established a lymphoblastoid cell line (MA214L) from the patient which maintains the translocation in culture. We have used Genethon microsatellite markers as sequence tagged sites (STSs) to isolate yeast artificial chromosome (YAC) clones to 5q and 6q from human genomic libraries. Using fluorescence in situ hybridisation (FISH) on metaphase preparations of MA214L, we have physically defined the translocation breakpoints between YAC clones on each chromosome arm. The genetic distance separating the flanking YACs on 6q21 is 3 cM, while that on 5q21 is 4 cM. To date this is the first report of these chromosomal regions being implicated in Wilms' tumourigenesis.